The fibroblast growth factor pathway serves a regulatory role in proliferation and apoptosis in the pathogenesis of intestinal atresia.

Abstract

Intestinal atresia occurs in 1:5000 live births and is a neonatal challenge. Fibroblast growth factor receptor 2b (Fgfr2b) is a critical developmental regulator of proliferation and apoptosis in multiple organ systems including the gastrointestinal tract (GIT). Fgfr2b invalidation results in an autosomal recessive intestinal atresia phenotype. This study evaluates the role of Fgfr2b signaling in regulating proliferation and apoptosis in the pathogenesis of intestinal atresia.
Wild-type and Fgfr2b-/- embryos were harvested from timed pregnant mice. The GIT was harvested using standard techniques. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) was used to evaluate apoptosis and bromodeoxyuridine to assess proliferation by standard protocols. Photomicrographs were compared (Institutional Animal Care and Use Committee-approved protocol 32-02).
Wild-type and mutant GIT demonstrate that deletion of the Fgfr2b gene results in inhibition of epithelial proliferation and increased apoptosis. Inhibited proliferation and increased apoptosis are specific to those tissues of normal Fgfr2b expression, corresponding to the site of intestinal atresia.
The absence of embryonic GIT Fgfr2b expression results in decreased proliferation and increased apoptosis resulting in GIT atresia. The regulation of proliferation and apoptosis in intestinal cells as a genetically based cause of intestinal atresia represents a novel consideration in the pathogenesis of intestinal atresia.

Full text

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The fibroblast growth factor pathway serves a
regulatory role in proliferation and apoptosis
in the pathogenesis of intestinal atresia
Timothy J. Fairbanksa, Frederic G. Salaa, Robert Kanarda, Jennifer L. Curtisa,
Pierre M. Del Morala, Stijn De Langhea, David Warburtona, Kathryn D. Andersona,
Saverio Belluscia, R. Cartland Burnsb,*
aDevelopmentalBiology Program, Division ofPediatric Surgery,Children’s HospitalLosAngeles, LosAngeles, CA 90027, USA
bDivision of Pediatric Surgery, University of Virginia Children’s Hospital, Charlottesville, VA 22908-0709, USA
Abstract
Background/Purpose: Intestinal atresia occurs in 1:5000 live births and is a neonatal challenge.
Fibroblast growth factor receptor 2b (Fgfr2b) is a critical developmental regulator of proliferation and
apoptosis in multiple organ systems including the gastrointestinal tract (GIT). Fgfr2b invalidation
results in an autosomal recessive intestinal atresia phenotype. This study evaluates the role of Fgfr2b
signaling in regulating proliferation and apoptosis in the pathogenesis of intestinal atresia.
Methods: Wild-type and Fgfr2b?/?embryos were harvested from timed pregnant mice. The GIT was
harvested using standard techniques.Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling)
was used to evaluate apoptosis and bromodeoxyuridine to assess proliferation by standard protocols. Pho-
tomicrographs were compared (Institutional Animal Care and Use Committee-approved protocol 32-02).
Results: Wild-type and mutant GIT demonstrate that deletion of the Fgfr2b gene results in inhibition of
epithelialproliferationandincreasedapoptosis.Inhibitedproliferationandincreasedapoptosisarespecific
to those tissues of normal Fgfr2b expression, corresponding to the site of intestinal atresia.
Conclusions: The absence of embryonic GIT Fgfr2b expression results in decreased proliferation and
increasedapoptosisresultinginGITatresia.Theregulationofproliferationandapoptosisinintestinalcells
as a genetically based cause of intestinal atresia represents a novel consideration in the pathogenesis of
intestinal atresia.
D 2006 Elsevier Inc. All rights reserved.
Intestinal atresia is a significant clinical problem facing
the neonatal patient population and can lead to significant
short- and long-term morbidities. The morbidity and
mortality of immediate surgical correction and delayed
manifestations of intestinal insufficiency are significant and
may lead to dependence on parenteral nutrition, infection,
loss of vascular access, and hepatic failure.
There are 2 classic theories of intestinal atresia. Tandler
[1], in 1902, proposed a lack of recanalization of the solid
cord stage of intestinal development as the cause of intestinal
0022-3468/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jpedsurg.2005.10.054
Presented at the 36th Annual Meeting of the American Pediatric
Surgical Association, Phoenix, AZ, May 29-June 1, 2005.
4 Corresponding author. Division of Pediatric Surgery, Surgery and
Pediatrics, UVA Children’s Hospital, PO Box 800709, Charlottesville, VA
22908-0709, USA.
E-mail address: cburns@virginia.edu (R.C. Burns).
Index words:
Intestinal atresia;
Fibroblast growth factor;
FGFR2B;
Proliferation;
Apoptosis
Journal of Pediatric Surgery (2006) 41, 132–136
www.elsevier.com/locate/jpedsurg

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atresia in the duodenum. Fifty-five years later, Louw and
Barnard [2] presented their observations on the origin of
intestinal atresia in the midgut. Observations of clinical
pathological specimens led to the hypothesis that intestinal
atresia was caused by an embryological bvascular accident.Q
Late-term embryonic canine pup mesenteric vessels were
ligatedinutero,andthepregnancieswereallowedtocontinue
to term. Two pups were found to have intestinal atresia after
ligation of the mesenteric vessels, providing proof of their
hypothesis. Although these 2 theories are classically accept-
ed, full understanding of this developmental defect is still
incomplete. Clinically,familialhereditaryformsofduodenal,
jejunoileal, colonic, and multiple atresias have been reported.
The associated presence of nongastrointestinal developmen-
tal defects raises the possibility of intestinal atresia being
a heritable condition in some cases [3-14].
The fibroblast growth factors act through tyrosine
kinase transmembrane receptors [15]. The fibroblast
growth factor receptor (Fgfr) gene family has genetic
linkage to skeletal dysplasias, autosomal dominant cranio-
synostosis syndromes, and mammary gland development
[16,17]. However, the Fgf receptor family has not been
implicated in gastrointestinal malformation in humans. We
have previously demonstrated Fgfr2b invalidation results
in a reproducible, autosomal recessive intestinal atresia
phenotype in mouse. The murine Fgfr2b null mutant
manifests cecal, colonic, and duodenal atresia [18-20].
Furthermore, intracardiac injection of india ink and studies
of angiogenesis in the developing colon have suggested
that this atresia is not the result of a vascular accident as
conventionally accepted [2,21].
The purpose of the current study is to determine the role
of proliferation and apoptosis in the pathogenesis of
intestinal atresia. Three processes that are essential to
organogenesis are proliferation, differentiation, and apopto-
sis. Growth factors and their receptors such as Fgfr2b
orchestrate the complex mesenchymal-epithelial cell inter-
actions that result in the organogenesis of complex
structures such the gastrointestinal tract. Homozygous
deletion of Fgfr2b in the previously described Fgfr2b?/?
knockout mutant murine model results in colonic atresia
with a complete penetrance and a uniform phenotype. This
phenotype is inherited in autosomal recessive fashion, as
part of a constellation of severe developmental defects. The
Fgfr2b?/?mutant permits evaluation of the role of this
signaling pathway in regulating proliferation and apoptosis
at critical time-points in development of the colon.
1. Materials and methods
1.1. Mutant embryos
A Cre-recombinase-mediated excision was used to
generate mice lacking Fgfr2b on the C57B1/6 murine strain
background as described by De Moerlooze et al [16]. The
Fgfr2b?/?mutant embryos are viable until birth, at which
time they have respiratory failure secondary to lung agenesis
[16,17]. The heterozygous (Fgfr2b+/?) mouse colony is
used to generate timed pregnancies for evaluation. In a
similar fashion, timed pregnancies of wild-type (Wt)
C57B1/6 littermates were used as controls. Embryos at
E10.5 (embryonic day 10.5 or 10.5 days after conception)
were removed from the uteri of pregnant mothers. These
specimens were then preserved in 4% paraformaldehyde,
embedded in paraffin, and sectioned sagittally at 8 lm using
a microtome (Leica, Bannockburn, IL). The sections were
processed and mounted for histochemical analysis.
1.2. Proliferation
Bromodeoxyuridine (BrdU; 0.2 mL; Amersham Bio-
sciences UK Limited, Bucks, England) was given via
intraperitoneal injection to Fgfr2b?/+(heterozygous) preg-
nant female mice at stage E10.5. The mice were euthanized
15 minutes after injection. The colon was dissected from
embryos and preserved in 4% paraformaldehyde. The BrdU-
labeled colon was dissected and oriented in a uniform
fashion for proper identification of structures. The sections
were then treated with monoclonal anti-BrdU (Clone BU-1)
RPN 202 as recommended by manufacturer (Amersham
Biosciences UK Limited). Vectashield was used as a
mounting medium (Vectashield, Burlingame, CA). The
sections were then photographed. To control for variability
in the uptake or absorption of BrdU, data are presented from
embryos of similar sizes from the same litter.
1.3. Apoptosis
The Wt and Fgfr2b?/?mutant colon sections were
harvestedinthetimedpregnantmannerdescribedpreviously,
then processed for evaluation of apoptosis. The In Situ Cell
Death Detection Kit, Fluorescein, kit for detection and quan-
tificationofapoptosisatsingle-celllevel,basedonlabelingof
DNA strand breaks (terminal deoxynucleotidyl transferase
biotin-dUTP nick end labeling [TUNEL] technology) from
Roche Applied Science (Indianapolis, IN) was used per
supplied instructions. Vectashield was used as a mounting
medium, and the sections were then photographed. Data are
presented from embryos of similar sizes from the same litter.
2. Results
2.1. Invalidation of the Fgfr2b gene results
in a marked downregulation of colonic
epithelial proliferation
Homozygous deletion of Fgfr2b results in colonic atresia
with complete penetrance and a uniform phenotype. This
phenotype is inherited in autosomal recessive fashion, as
part of a constellation of severe developmental defects. At
stage E10.5, the Fgfr2b?/?mutant colon and that of the
The fibroblast growth factor pathway 133

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Fgf10?/?mutant are grossly phenotypically equal and only
the Fgfr2b?/?data are shown. The pathogenic event of
intestinal atresia is yet to be detectable by simple micros-
copy. The colon of the Wt and Fgfr2b?/?mutant at stage
E10.5 were dissected, preserved, and sectioned as described.
The sections were then treated an anti-BrdU detection kit.
Cells staining red have taken up BrdU, which is detected by
the anti-BrdU antibody, indicating proliferation at the time
of BrdU exposure (Fig. 1A and B). The Vectashield stains
all other cells blue. The photomicrographs in Fig. 1
demonstrate that epithelial proliferation is severely restricted
in the Fgfr2b?/?mutant colon.
There is a marked decrease in the number of proliferating
cells in the Fgfr2b?/?mutant as compared with the Wt
(Fig. 1A and B). There is also a decrease in the rate of
proliferation of the colonic mesenchymal cells in the
Fgfr2b?/?mutant as compared with its Wt littermate
(Fig. 1A and B). The epithelium of the Wt mouse
proliferates at a similar rate to the surrounding mesenchyme
(Fig. 1C). This is in stark contrast to the epithelium of the
Fgfr2b?/?mutant. With loss of Fgfr2b function, there is
near-complete absence of epithelial proliferation, whereas
mesenchymal cells immediately adjacent to the epithelium
continue to proliferate (Fig. 1D, white arrow). The effect of
deletion of Fgfr2b is a decrease in the rate of proliferation in
both the mesenchyme (to a lesser degree) and in the
epithelium (nearly complete).
2.2. Invalidation of the Fgfr2b gene results in
stimulation of colonic epithelial apoptosis
The colons of the Wt and Fgfr2b?/?mutant at stage
E10.5 were dissected, preserved, and sectioned as described.
The sections were then treated with the In Situ Cell Death
Detection Kit, Fluorescein, to detect TUNEL. The sections
of Wt and Fgfr2b?/?mutant are shown in Fig. 2. The cells
staining green are in the process of apoptosis as detected by
TUNEL. The Wt colon shows very few apoptotic cells
(Fig. 2A), whereas the Fgfr2b?/?mutant shows a dramatic
increase in the rate of apoptosis (Fig. 2B). Furthermore, the
pattern of apoptosis is essentially exclusive to the epithelium.
This occurs while the mesenchymal cells of the Fgfr2b?/?
mutant immediately adjacent to the epithelial cells show very
few apoptotic cells. The magnified view focusing on the
epithelial cells (white arrows, shown in Fig. 2C and D)
further demonstrates and confirms the cell-specific distribu-
tion of apoptosis in the Fgfr2b?/?mutant, whereas the Wt
specimen confirms near absence of apoptosis.
3. Discussion
Intestinal atresia has classically been understood to be the
result of a vascular accident or insufficiency. Our interest in
the heritable causes of developmental defects (including
intestinal atresia) led us to evaluate the regulatory role of the
Fgf10 and Fgfr2b signaling pathway in the manifestation of
intestinal atresia. We have previously shown that the
vascular structures at the site of intestinal atresia are present
and patent [21]. Despite the normal vascular development,
the atresia occurs in a reliable heritable fashion in these
2 mutant mice. The Fgf10-Fgfr2b signaling pathway is
known to regulate mesenchymal-epithelial interactions, and
its loss of function results in gastrointestinal tract atresias
without vascular malformation, raising questions about the
Fig. 1
ing cells of colon at E10.5 demonstrates strong proliferation in both
the mesenchyme and epithelium of the Wt colon (A) and decreased
proliferation in the Fgfr2b?/?mutant (B). High-power magnifica-
tion of both defines the epithelial proliferation in Wt (C) and clear
absence of epithelial proliferation in the Fgfr2b?/?mutant, despite
some preservation of mesenchymal proliferation (D).
Proliferation. Bromodeoxyuridine labeling of proliferat-
Fig. 2
apoptosis in Wt and Fgfr2b?/?mice. The Wt colon displays very
little apoptosis at this developmental stage (A), whereas the mutant
colon (B) has strong evidence of apoptosis at the same stage. High-
power magnification further defines the cell-specific regulation of
apoptosis with negligible findings in the Wt mouse (C), whereas a
stark contrast is present with abundant apoptosis restricted to the
epithelium of the Fgfr2b?/?mutant (D).
Apoptosis. TUNEL assay of colon at E10.5 demonstrates
T.J. Fairbanks et al.134

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role of vasculogenesis in intestinal atresia. Little is currently
known about the regulation of proliferation and apoptosis in
the developing intestine, and our data indicate a critical role
for the Fgf10-Fgfr2b signaling pathway. Clearly, in the loss
of function of this pathway, a failure of proliferation and
potentiation of apoptosis occurs at the site of intestinal
atresia, giving a logical alternative explanation of the
pathogenesis of the atresia. This consideration of a
regulatory mechanism balancing proliferation and apoptosis
in the developing intestine questions the classically accepted
vascular theories of intestinal atresia. With the current
evidence demonstrating failure of proliferation and in-
creased apoptosis in the atretic mutant colon, new oppor-
tunities arise for investigating the pathobiology of intestinal
atresia and may lead to opportunities to create novel
therapeutic alternatives.
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Discussion
Allan Goldstein, MD (Boston, MA): You show that there is
increased apoptosis and less cell proliferation. Is that true
along the entire length of the GI tract, and if so, why does
the atresia occur only in the proximal colon? Can you
explain that?
Timothy Fairbanks, MD (response): Actually, we did look
at the rest of the gastrointestinal tract, and the specimens
we showed were specific to the colon and this is the part
of the colon which undergoes the atresia. If we looked at
the cecum or the small intestine immediately adjacent,
the apoptosis is not upregulated. This is a tissue-specific
event happening in the same spot where the atresia will
develop, not across the whole gastrointestinal tract.
Jean-Martin Laberge, MD (Montreal, Quebec, Canada): I
guess the main question is whether our patients are
mutants. Maybe some are, but I think most of them are
not. Can you actually test for these receptors and factors
in babies? Maybe you could start with the familial cases
who have multiple atresias.
Timothy Fairbanks, MD (response): An excellent point. It is
unlikely that the FGF-10, the gene we are talking about
today, is the cause of most of the intestinal atresias we
see because the phenotype is so severe. These animals
have pulmonary agenesis, multiple intestinal atresias, and
cleft palate. This is a severe defect. There could be either
a point mutation to a gene like FGF-10 or a similar gene
which acts in the jejunum or the ileum, and a point
mutation or a somatic mutation could be the cause of the
intestinal atresias we see clinically rather than a knockout
model similar to the one we have looked at here.
Kerry Bergman, MD (Summit, NJ): This is a very elegant
study that gives good data to support the presence of
webs and cords, but how does your data then jive with
the type 3 and type 4a atresias?
Timothy Fairbanks, MD (response): It is interesting to note
that the colonic atresia is present in 100% with a uniform
phenotype, with the complete penetrance, but the same
FGF-10 and the FGFR2b knockout mouse has a
duodenal atresia phenotype where we see a 30%
The fibroblast growth factor pathway 135

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penetrance with a complete variability of phenotype
where we will see a web, we will see a completely
normal duodenum, or we will see a complete atresia. Our
understanding of what is causing the absence of the same
gene to cause a web vs a complete atresia in one animal
to the next is incomplete at this stage. As to why you
knock out the same gene and you will get a different
phenotype within the same organ, we just have more to
learn. Also, I believe likely some of these intestinal
atresias are the result of a spontaneous occlusion of a
mesenteric vessel as classically described and others are
developmental defects like the ones we are talking about.
Edmund Yang, MD, PhD (Nashville, TN): Could you review
the expression patterns of FGF-10 and the receptor in
normal mice and humans?
Timothy Fairbanks, MD (response): FGF-10 is expressed in
the distal colon most intensely within the rectum. It is
also expressed in the cecum and duodenum as well as
the developing lung. We do have some data published
which show the expression pattern of FGF-10 along
with several of its upstream and downstream regula-
tory molecules.
Patricia Donahoe, MD (Boston, MA): I applaud your use of
the knockout animals to direct us to the genetics of these
defects. We need to appreciate and use this approach
more broadly as a society.
I wonder, since this is such a strong positional effect,
whether there is some interaction with the HOX genes or
the PAX genes.
Timothy Fairbanks, MD (response): We still have more
work to do to determine what are the upstream and
downstream regulators of the FGF-10 pathway. The
effect of the knockout of FGF-10 is very severe, and it is
possible that some of the downstream or upstream
regulators would have a less severe phenotype which
would be more similar to the phenotype we see in the
infants that are born.
Marshall Schwartz, MD (Philadelphia, PA): How does
your model explain what we observe clinically? Your
studies have shown a complete separation of the
bowel, including muscle absence and vascular inter-
ruption. The receptor that you have described is
located in the mucosa, and thus, how does this
location explain the loss of muscle tissue, serosa, and
blood vessels?
Timothy Fairbanks, MD (response): One of the things we
found with the knockout models of the FGF-10 or
FGFR2b gene is 2 separate animal models have the same
or similar phenotype. These are a complete knockout of
the genes. We also have a hypomorphic model where we
have a decreased activity of FGF-10. In that hypomor-
phic model, the colon that we see is probably 75% the
length of a normal colon, and when you section that
colon, you will find that in the proximal colon the
epithelial and the muscular layers are all normal. If you
look at what would be the transverse colon, you start to
see abnormalities of the villi and you will see the
thinning of the muscular layers, and when you get all the
way to the terminal end point of the atresia, you will see
severe abnormalities both of the epithelial layer and of
the muscular layers. I am not sure why the down-
regulation of this gene causes a different phenotype in the
muscular layers and the epithelial layers when it is
downregulated as opposed to when it is completely
knocked out. There is clearly much more to learn about
this and other models of intestinal atresia.
T.J. Fairbanks et al. 136