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Failure of intestinal adaptation (IA) in short bowel syndrome
(SBS) can lead to significant morbidity and mortality. We have established a
high throughput, low-cost model of SBS in zebrafish that reproduces the
pathophysiology and IA seen in human patients with SBS. In the SBS zebrafish
model we observe maximum weight loss, peak intestinal epithelial cell
proliferation, and other markers of adaptation at 2 weeks. Although the exact
mechanisms of IA are unknown, we note a marked increase in genes of the Hippo
pathway and the Wnt/ ?-catenin pathway at the time of peak epithelial proliferation.

Regulation of the downstream Hippo transcription factor Yes-associated protein
(YAP) is crucial in modulating the development of organ size and
structure. Preliminary work in our laboratory revealed an increase in

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proliferating cells in the intervillus pocket (homologous to
intestinal crypts) in SBS proximal intestine at two weeks compared to sham and
diverted distal SBS intestine. In addition, we identify an increase in
cytoplasmic YAP expression and a key Wnt modulator, ?-catenin in the
intervillus pockets. It is known that YAP is crucial in the degradation of
cytoplasmic ?-catenin in the Wnt OFF state. In the Wnt ON state, ?-catenin
is not destroyed and is allowed to enter the nucleus and influence
transcription. Therefore, the central aim of this proposal is to determine the
timing of the crucial signaling mechanisms within the YAP/Hippo
pathway that modulate canonical Wnt/?-catenin signaling in
IA in zebrafish short bowel syndrome that will provide novel therapeutic
targets for small molecules for enhancing IA in patients affected by
SBS.

 

 

Failure of intestinal adaptation (IA) in short bowel syndrome
(SBS) can lead to high morbidity and even death. In children, necrotizing
enterocolitis, volvulus, and gastroschisis are devastating conditions that can
result in massive intestinal resection leading to SBS 1. SBS leaves the
patient with an insufficient amount of intestine necessary to absorb enough
nutrients for survival. IA fails in many patients2. It is a poorly understood
process that is characterized by structural and functional changes (increased mucosal
absorptive surface area and digestive capacity) that occur in the remaining
intestine after a significant portion of intestine is resected (approximately
70%). No therapies exist that efficiently rescue the failure of IA. Current
therapies leave patients dependent on IV nutrition, which can further
intestinal atrophy, complications of central venous line sepsis, liver failure,
and poor quality of life. Intestinal transplantation requires life-long
immunosuppressive therapy and has a 3-year survival rate of 40%1.

 

The cost of care per child with SBS over a 5-year period is $1,
619,851 3. SBS studied in murine animal models are expensive, require long
experimental time points, and have high mortality rates. Zebrafish (Danio
rerio) are relatively inexpensive, have intestinal function analogous to
humans, and have a high survival rates after SBS surgery (approximately 90%).

Our laboratory has established a zebrafish model of SBS that demonstrates IA
with many of the components identified in human patients. The goal of our
proposal is to identify key molecular targets that enhance IA in an SBS model
that abrogate the morbidity and mortality in human SBS patients.

 

This proposal is designed to evaluate the interaction of the
YAP/Hippo and canonical Wnt/?-catenin signaling
pathways at the level of proliferating stem cells in the intervillus pockets of
zebrafish intestine undergoing intestinal adaptation. Recent studies have revealed the
Hippo pathway to be important in non-homeostatic states4,
whereas the canonical Wnt pathway is important in both intestinal homeostasis
and regeneration5. Our zebrafish model is novel and recapitulates the SBS
phenotype and IA that occurs in humans, and is characterized by significant
weight loss, increased villus epithelial

perimeter (VEP), inner epithelial perimeter (IEP), and stem cell
proliferation. Zebrafish are less costly compared to murine models, have
shorter operative times (approximately 5 minutes versus 30 minutes in murine),
a higher survival rate, and are easier to maintain compared to other SBS animal
models.

 

Furthermore, genome wide gene expression data in zebrafish
intestine indicate function analogous to human small and large intestine 6.

The zebrafish intestine consists of 7 segments in which segment 1-5 are
homologous to human small intestine. The distal two segments (S6 and S7) are
phenotypically similar to cecum and colon7. Previous studies in our lab have
shown peak epithelial stem cell proliferation (measured as BrdU positive cells
per hemivillus) in the intervillus pockets of proximal SBS bowel at the 2-week
time point7. RNA sequencing of zebrafish intestine undergoing IA demonstrate that
genes both upstream and downstream of the YAP/Hippo and Wnt/?-catenin pathway were
increased by 2-4-fold at 2 weeks. It has been established that both the Wnt/ ?-catenin and
YAP/Hippo pathways influence intestinal stem cell proliferation. A key factor
affected by both pathways is ?- catenin.

In the Wnt OFF state, ?-catenin is degraded in the cytoplasm by a degradation complex regulated
by YAP; however, the precise mechanism of degradation remains unknown. Exposing
the interactions between the Wnt/ ?-catenin and
YAP/Hippo pathway in the proliferating stem cells during IA will help us
uncover potential therapeutic targets to enhance intestinal adaptation that
will lead to a decrease in the morbidity and mortality associated with short
bowel syndrome.

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