Articles in PresS. Am J Physiol Gastrointest Liver Physiol (February 26, 2015). doi:10.1152/ajpgi.00158.2014

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Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in

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the adult mouse small intestine

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Denise Al Alam1,2,&, Soula Danopoulos1,2, Kathy Schall1,2, Frederic G Sala1,2, Dana

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Almohazey1,2, G. Esteban Fernandez1, Senta Georgia2, Mark R Frey1,2, Henri R Ford1,2,

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Tracy Grikscheit1,2,*, Saverio Bellusci1,2,3,4*

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Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA Developmental Biology and Regenerative Medicine Program, Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA Department of Internal Medicine II. University of Giessen Lung Center and Member of the German Lung Center. Member of the DZL. Klinikstrasse 36, 35392 Giessen. Germany Institute of Fundamental Medicine and Kremlevskaya St 18, Kazan, 420008, Russia.

Biology,

Kazan

Federal

University,

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&

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email: [email protected]

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Address: Saban Research Institute, 4661 W Sunset Blvd, Los Angeles, CA, 90027

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Key words: Fgf10, Fgfr2b, small intestine, differentiation

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* These authors contributed equally

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Running title: Fgf10 in small intestine

Correspondence to : Denise Al Alam, PhD

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1 Copyright © 2015 by the American Physiological Society.

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Abstract

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Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration,

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proliferation, differentiation and apoptosis. Intestinal epithelial cells consist of absorptive

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and secretory lineage. The latter is comprised of goblet, Paneth and enteroendocrine

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cells. Fibroblast Growth Factor 10 (FGF10) plays a central role in epithelial cell

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proliferation, survival and differentiation in several organs. The expression pattern of

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FGF10 and its receptors in both human and mouse intestine and their role in small

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intestine have yet to be investigated. First, we analyzed the expression of FGF10,

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FGFR1 and FGFR2, in the human ileum and throughout the adult mouse small intestine.

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We found that FGF10, FGFR1b and FGFR2b are expressed in the human ileum as well

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as in the mouse small intestine. We then used transgenic mouse models to overexpress

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Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in

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the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in

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vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10

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decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2 and Sox9. FGF10

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inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell

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differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10

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overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2

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double positive cells, suggesting that goblet cells replace Paneth cells. Further studies

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are needed to determine the mechanism by which Fgf10 alters cell differentiation in the

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small intestine.

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Introduction

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The intestine is lined by a single layer of epithelial cells that allows the exchange of

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nutrients but also functions as a physical barrier against bacteria and ingested toxins.

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The intestinal epithelium self-renews continuously while maintaining this barrier. This

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renewal relies on the right balance of cell proliferation, migration, differentiation and

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apoptosis. Stem cells near the base of the crypt of Lieberkühn divide, feeding into a

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transit amplifying (TA) population. Differentiating cells migrate out of the TA

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compartment either into the villi or the base of the crypt. Fully mature intestinal epithelial

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cells (IECs) belong to either the absorptive or the secretory cell lineage. Secretory cell

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types are goblet, Paneth, tuft and enteroendocrine (27). Secretory lineages arise from

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progenitors expressing Atonal Homolog 1 gene (Atoh1, also called Math1) (37, 44, 48).

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The notch effector, hairy/enhancer of split 1 (Hes1), deletion of which results in

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excessive formation of all three secretory cells types, inhibits Atoh1 expression (20, 40,

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44). Within the secretory lineage, enteroendocrine cell fate specification depends on the

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expression of Neurogenin 3 (Neurog 3) (19, 26); Paneth cell differentiation and

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maturation rely on the expression of SRY-box containing gene (Sox9) and Wnt signaling

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via Frizzled 5 (1, 27) and differentiation of goblet cells requires Kruppel like factor 4

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(Klf4) (22). SAM pointed domain containing Ets transcription factor (Spdef) regulates

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both goblet and Paneth cell differentiation. Deletion of Spdef severely disrupts the

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maturation of goblet and Paneth cells (13) while overexpression of Spdef in mice

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increases goblet cell differentiation and decreases Paneth cells, enterocytes and

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enteroendocrine cells (28).

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Fibroblast growth factor 10 (FGF10), one of 22 members of the FGF family, is known to

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play a central role in cell proliferation and/or differentiation of the epithelium in several

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organs (2, 34, 39, 46). During development of the gastrointestinal tract, Fgf10 is

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expressed in the mesenchyme of the stomach, duodenum, cecum and colon (4, 9, 33)

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and is critical for the development of these organs (4, 29, 33, 41, 42). The loss of Fgf10

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in mice results in duodenal, cecal, and colonic atresia (8, 10, 11, 21). We recently

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showed that Fgf10 expression is induced in the ileum of mice during gut adaptation (41).

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Moreover, Fgf10 overexpression promotes the formation of tissue-engineered small

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intestine (42). However, to date, the impact of gain or loss of Fgf10 signaling on adult

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mouse small intestine has not been investigated.

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In this study, we analyzed the expression of FGF10, its receptors FGFR1 and FGFR2 as

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well as other FGFR2 ligands in the human ileum and the three segments of the adult

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mouse small intestine (duodenum, jejunum and ileum). We showed that FGF10,

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FGFR1b and FGFR2b are expressed in the human ileum. In the mouse intestine, Fgf10

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is expressed in the duodenum, while Fgfr1 and Fgfr2 are expressed throughout the

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intestine. Furthermore, we demonstrated that overexpression of Fgf10 both in vivo and

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in vitro induced goblet cell differentiation and reduced Paneth cells, while sequestering

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Fgfr2b ligands with a soluble receptor did not affect intestinal differentiation. Moreover,

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FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2 and Sox9 in ileal

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enteroids cultured in vitro. FGF10 inhibited Hes1 expression in the enteroids, suggesting

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that FGF10 induces goblet cell differentiation likely through the inhibition of Notch

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signaling. Interestingly, Fgf10 overexpression in vivo increased the number of goblet

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cells in the crypt compartment. Furthermore, we showed that Fgf10 overexpression for 3

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days in vivo and in vitro increased the number of Mmp7/Muc2 double positive cells.

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Taken together, these results suggest that goblet cells replace Paneth cells following

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Fgf10 overexpression. We demonstrated that Fgf10 plays an important role in intestinal

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cell differentiation. Further studies are needed to determine the mechanism(s) by which

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Fgf10 alters cell differentiation in the small intestine.

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Materials and Methods

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Human subjects

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Fresh human tissue was obtained from patients 3 months -18 years old, admitted for

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surgery at Children’s Hospital Los Angeles under an IRB-approved protocol to collect

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waste tissue derived from surgeries that is not needed for pathologic diagnosis. Families

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sign consent for the tissue collection and demographic and curated medical history data

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are available through the protocol. The indications for surgery for these patients did not

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include primary intestinal disease.

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Mice

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All the mice were housed in the Animal Care facility of the Saban Research Institute,

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Children’s Hospital Los Angeles. The Institutional Animal Care and Use Committee

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approved all animal protocols used in this study in strict accordance with the

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recommendations in the Guide for the Care and Use of Laboratory Animals of the

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National Institute of Health. The approval identification number for Children’s Hospital

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Los Angeles is AAALAC A3276-01. CD1 Wild type mice were purchased from the

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Charles Rivers Laboratory and C57Bl/6 mice from the Jackson Laboratory.

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Generation of mutant mice

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For this study we used mice that allow ubiquitous, inducible, and reversible

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overexpression of Fgf10 as well as a soluble form of Fgfr2b (sFgfr2b) as previously

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described (6, 14, 30, 32). Briefly, mice (CD1 mixed background) expressing rtTA under

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the ubiquitous Rosa26 promoter were crossed with lines harboring tet(O)sFgfr2b or

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tet(O)Fgf10 to obtain animals carrying both transgenes (R26rtTA/+; tet(O)sFgfr2b/+ and

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R26rtTA/+; tet(O)Fgf10) and single transgene littermate controls (all mice carried only a

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single copy of a given transgenic allele). Tet promoter-induced Fgf10 overexpression

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was achieved by feeding doxycycline (dox)-containing food (Rodent diet with 0.0625%

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Doxycycline, Harlan Teklad TD01306) to double transgenic adult (4 week-old) mice

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(R26rtTA/+; tet(O)Fgf10/+) for a period of 10 days; sFgfr2b induction was achieved by

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feeding doxycycline to double transgenic adult (4 week-old) mice for 1 month or 3

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months. Double transgenics without dox or single transgenics with or without dox were

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used as controls as described in Results. At the end of the dox treatment, mice were

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euthanized and the small intestines were harvested and separated in segments

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(duodenum, jejunum and ileum). Tissues were either fixed in formalin or frozen in liquid

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nitrogen for RNA extraction.

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Beta-galactosidase staining

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Mlcv1v-nLacZ-24 or Fgf10LacZ reporter mouse was used to investigate the spatio-

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temporal expression of Fgf10 throughout the small intestine (23, 24). Small intestines

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were collected from 4-week old Fgf10LacZ animals and stained for beta-galactosidase

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activity as previously described (38). Briefly, samples were shortly fixed in 4% PFA, then

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stained in a LacZ solution containing a final concentration of 2 mg/mL of X-gal overnight

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at 37˚C (rpi research products). These samples were then fixed, paraffin-embedded,

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sectioned at 5μm and mounted on slides. Slides were deparaffinized, re-hydrated in an

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ethanol gradient, counter-stained with nuclear fast red, dehydrated, cleared with

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histochoice, and mounted with Permount (Fisher Scientific, Fair Lawn, New Jersey).

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Immunohistochemistry

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All three segments of the small intestine (duodenum, jejunum and ileum) were collected,

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fixed and paraffin embedded for histological analyses. The samples were sectioned at 5

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m, then deparaffinized and re-hydrated. The slides were stained with Hematoxylin and

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Eosin to examine their histology. The depth of the crypts and height of the villi were

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measured using image J software (National Institutes of Health, Bethesda, MD). Alcian

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blue staining was used to visualize goblet cells. Immunohistochemistry for FGFR1 and

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FGFR2 was performed using the following antibodies: rabbit anti-FGFR1 (1:100, Flg (C-

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15) Santa Cruz), rabbit anti-FGFR2 (1:200, Bek (C-17) Santa Cruz) and anti-rabbit IgG

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(1:100, Santa Cruz). For these antibodies, an antigen retrieval step was performed by

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boiling the slides in a microwave for 12 min in Tris-EDTA (10 mM Tris Base, 1mM EDTA

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Solution, 0.05% Tween 20, pH = 9.0). To confirm the stainings, we also used rabbit anti-

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FGFR1 (1: 100, Abcam ab63601) and rabbit anti-FGFR2 (1:100, Abcam ab10648) with

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antigen retrieval steps as recommended by the manufacturer. For the remaining

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immunohistochemistry (IHC) and immunofluorescence (IF) stainings, the slides were

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boiled in the microwave for 12 min in 10mM sodium-citrate buffer pH=6.0. Cell

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proliferation was assessed by IHC using mouse anti-proliferating cell nuclear antigen

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(PCNA) (1:100, Vector Laboratories) and IF staining for Phh3 (1:100, cell signaling). The

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staining was visualized using Dako cytomation kit following the manufacturer’s

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instructions. Slides were dehydrated and mounted with DPX. IF staining was performed

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to identify Paneth cells (anti-Lysozyme 1:100, Dako) and enteroendocrine cells (rabbit

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anti-chromogranin A (CGA) 1:100, Abcam). Cell death was assessed using rabbit anti-

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Caspase 3 (active) (1:500, R&D Systems). Cy3-conjugated secondary antibodies were

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used for the IF stainings. Slides were counterstained DAPI (1: 500, Life Technologies)

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and mounted using ProLong Diamond Antifade Mountant (Life Technologies). Images

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were acquired using a camera attached to an upright fluorescent microscope (Leica

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DM5500).

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Cell counting and quantification

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Epithelial nuclei, lysozyme and Chromogranin A stained cells were counted semi-

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automatically using MetaMorph 7.7.3.0 software (Molecular Devices, Sunnyvale, CA).

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First, the blue (DAPI) channel was processed as follows: background was subtracted by

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manual thresholding, intensity was normalized by dividing by a 20×20 low-pass filtered

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copy of the channel, and the normalized nuclei were smoothed with a 5×5 median filter

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to preserve edges. A binary image of the smoothed nuclei was created by thresholding

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automatically using the isodata histogram algorithm under visual inspection to manually

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adjust the threshold value if needed. Overlapping binary nuclei were separated by

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Watershed segmentation using FoveaPro 3.0 software (Reindeer Graphics, Asheville,

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NC). To count Lysozyme and Chromogranin A positive cells, the red (Cy3) channel was

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processed in the same manner as the blue channel except that the thresholding step

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employed the "legacy heuristic" algorithm and an additional step to fill all dark holes

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(where nuclei didn’t show in Blue) was added just prior to the segmentation.

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The total number of PCNA, Alcian blue, Lysozyme and Chromogranin A positive cells

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and the total number of epithelial cells and/or epithelial cells per crypt were counted

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separately for 3 randomly selected high power fields (HPF=20x magnification) per

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sample per mouse and averaged, with six mice in every experimental set. The total

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number of cell-type positive cells per the total number of epithelial cells, or percent cell-

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type positive was calculated for each sample. Results were reported as percent ± SEM.

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Mouse Crypt cultures

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Mouse crypt culture were isolated and grown from wild type mouse ileum as previously

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described (35). Briefly, 6 cm of mouse ileum were isolated, washed and treated with 2

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mM cold EDTA for 30 min at 4C. The suspension is transferred into Sucrose:sorbitol

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buffer, gently shaken until the crypts start separating and strained through 70 m filters.

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After washing and centrifugation, the crypt units were embedded in matrigel containing

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EGF (50 ng/ml), Noggin (10 ng/ml) and R-Spondin (500 ng/ml) and incubated at 37C to

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allow the polymerization of the matrigel. Culture media containing DMEM/F12, L-

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Glutamine, Penicillin/Streptomycin, Hepes (10mM), N2 and B27 supplement was added

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to the matrigel. The crypts were grown for 7 days then passaged by gently dissociating

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the matrigel and breaking down the enteroids, then plated and cultured for 3 days before

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treatment for 4 days with 200 ng/ml of human recombinant FGF10.

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Crypts from Rosa26rtTA, Tet(O)Fgf10 animals were isolated as described above,

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replacing 1mM cold EDTA with 3 mM EDTA. Enteroids were treated with 2 g of

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doxycycline for 72 hours, after which the enteroids from the same experiment were

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pooled, split for RNA extraction and histology. RNA was extracted for gene expression

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analyses.

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Enteroid histology

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Enteroids fixed in 4% PFA were then embedded in histogel, dehydrated through ethanol

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gradient, embedded in paraffin, and sectioned for histology. IF staining was performed

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using anti-MMP7 antibody (1:100, Vanderbilt University), anti-Muc-2 (1:100, Santa Cruz),

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anti-Lysozyme (1:100, Dako), anti-Phh3 (1:100, cell signaling), and anti-E-cadherin

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(1:200, BD Biosciences). Slides were visualized under a Leica DM5500. Cells were

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counted in two images per sample in 4 independent experiments from 4 independent

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animals at 20X magnification and results are reported as percentage of positive cells to

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the total number of cells (nuclei).

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Whole Mount Mouse enteroid staining

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The mouse enteroid cultures in polymerized matrigel were rinsed with PBS and fixed for

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30 minutes in 2% PFA. The fixation was then quenched with 50 mM NH4Cl for 30

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minutes at 4°C. Crypts were permeabilized using 0.05% Triton X-100 in PBS for 30

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minutes. Blocking was achieved using 3% BSA in PBS for at least one hour at room

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temperature. Cell proliferation was assessed using phospho-histone H3 antibody (1:100,

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Cell Signaling), in which the cultures were incubated overnight at 4°C. The staining was

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visualized using Alexa Fluor® 647 Donkey anti-Rabbit (1:100, Life Technologies), in

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which the cultures were incubated overnight at 4°C. Nuclei were stained using 2μg/mL

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Hoechst 33342 fluorescent stain (Thermo Scientific) for 20 minutes. Cultures to

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ultimately be suspended in PBS when imaging.

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Z-stack images were acquired with an LSM 700 confocal system mounted on an

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AxioObserver Z.1 inverted microscope equipped with a 20x/0.8 Plan-APOCHROMAT

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lens and controlled by ZEN 2009 software (Carl Zeiss Microimaging, Thornwood, NY).

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Fluorescence images of DAPI and Alexa Fluor® 647 were obtained using excitation

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laser lines of 405 and 639 nm and emission filters of short-pass 490 nm and long-pass

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640 nm, respectively. The z-slice interval was 5 μm with the confocal pinhole set to 1

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Airy unit for Alexa Fluor® 647. A z-correction factor of 1.33 was used to obtain more

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accurate z scaling. If needed, the laser transmission percentage and/or detector gain

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were increased linearly with depth to obtain more uniform brightness (z-brightness

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correction). To measure the total visualized volume of each specimen the DAPI and

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Alexa Fluor® 647 channels were first combined using the Image Calculator MAX

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function of Fiji ImageJ software (36). Then, volume measurements were made with

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Imaris software by creating Surface objects using manual thresholding (Bitplane AG,

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Zurich).

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RT-PCR and qRT-PCR analyses

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RNA was isolated from adult wild type (C57Bl/6) mouse small intestines (duodenum,

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jejunum, and ileum) (n=3) and E14.5 wild type whole mouse embryo (n=3). One

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microgram of RNA was used for cDNA synthesis using tetro cDNA synthesis kit (Bioline).

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RT-PCR reactions were performed following the manufacturer’s instructions using either

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the Taq PCR Master Mix (Qiagen, Valencia, CA) and using the primers previously

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described and detailed in table 1 (38). Human primers used are as follows: FGF1 (L:

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CCTCGGCCTACAAGCTCTTT;

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TGGAGAACAGCGCCTACAGT;

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GCGCTCACACACAGAGAGAA; R: CAGCAGAATCATAATTGTTTCCAT), FGF10 (L:

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GAAGGAGAACTGCCCGTACA; R: GGCAACAACTCCGATTTCTACT), FGF22 (L:

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TTCTCCTCCACTCACTTCTTCC; R: CACGTGTACAGAGCGGATCTC), FGFR1b (L:

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GCATTCGGGGATTAATAGCTC; R: CCACAGGTCTGGTGACAGTG), FGFR2b (L:

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GCACAAGCTGACCAAACGTA; R: CTGGACTCAGCCGAAACTGT), and CDH1 (L:

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TGGAGGAATTCTTGCTTTGC; R: CGCTCTCCTCCGAAGAAAC).

R: R:

AGAGGAGTTTGGGCTTCTTGTA),

FGF3

(L:

GGAGAAGAGACCCCTGATGG),

FGF7

(L:

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Beta-galactosidase staining of whole mount mouse enteroid culture

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Enteroids from Axin2-LacZ animals were isolated as previously described, using 2mM

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EDTA. Enteroids were treated with 200ng/ml FGF10 for 48 hours, with media change

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and FGF10 supplementation occurring at 24 hours. Mouse enteroid cultures were

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stained for beta-galactosidase activity whilst in matrigel. Samples were briefly washed in

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PBS and fixed 2% PFA (2 minutes). They were then momentarily washed in LacZ buffer

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and stained in a LacZ solution containing 2 mg/mL of X-gal overnight at 37˚C (rpi

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research products).

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Statistical Analysis

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Statistical analyses were performed using Graphpad Prism software. Paired t tests

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compared the results from the enteroids cultures. Data are presented as average values

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± S.E.M. The results were considered significant if p0.05.

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Results

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Expression of FGF10 and its receptors FGFR1 and FGFR2 in human ileum

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Little is known about the expression of FGFs in human intestine. We first sought to study

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the expression of FGFR1, FGFR2 and FGF10 family members (FGFR2b ligands): FGF1,

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FGF3, FGF7 (also known as keratinocyte growth factor or KGF), FGF10 and FGF22 in

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healthy human ileum tissue obtained from patients aged 3 months -18 years old who

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required surgery for various indications that did not include primary intestinal disease. In

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most cases, these patients required short-term intestinal diversion 6+ weeks prior to

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tissue collection and were assessed as healthy and nutritionally replete at the time of

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surgery to reconnect the intestine. Hematoxylin and Eosin staining of human ileum show

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normal crypt and villi (Figure 1A-B). Staining by immunohistochemistry, using Flg (C-15)

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antibody, demonstrated that FGFR1 is expressed in human ileum throughout the length

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of the crypt and villi (Figure 1 C). This distribution was confirmed using a second FGFR1

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antibody from Abcam (Figure 1D). The use of matching rabbit IgG as primary antibody

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did not show any non-specific staining (Figure 1 E). Moreover, the Flg C-15 blocking

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peptide competed away binding, supporting the specificity of this antibody (Figure 1E’).

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FGFR2 staining, using Bek (C17) antibody, was also seen throughout the epithelium of

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the ileum and to a lesser extent in the mesenchyme (Figure 1F). This pattern was

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confirmed using a second FGFR2 antibody from Abcam (Figure 1G). Matched IgG

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primary and peptide competition staining confirmed the specificity of staining (Figure 1H-

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H’).

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To determine the expression of the ligands of FGFR1 and FGFR2 in the human ileum,

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we performed qRT-PCR on the same samples. We detected FGF7 and FGF10 in the

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human ileum but not FGF1, FGF3 and FGF22 (Figure 1I). We defined the threshold for

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positive expression at or before 35 RT-PCR cycles. Similar to protein expression, both

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FGFR1b and FGFR2b receptor genes were expressed in the human ileum as shown

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(using E-cadherin as reference, since b isoforms of these receptors are expressed

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exclusively in the epithelium) (Figure 1J).

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Expression of Fgf10 family members and Fgf10 receptors in the adult mouse small

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intestine

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Next, we aimed to assess the expression of the Fgf10 family members, as well as Fgf10

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receptors Fgfr1 and Fgfr2, in the three segments of the adult mouse small intestine.

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Analyzing the Fgf10LacZ reporter mouse, we found that Fgf10 is strongly expressed in the

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mesenchymal compartment of adult mouse duodenum, but not detected in the jejunum

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or the ileum (Figure 2A, n=3). Fgf10+/+ (LacZ-negative) control littermates did not show

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any LacZ staining (n=3). In order to confirm that Fgf10 was expressed only in the

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mesenchyme, we separated epithelium from the rest of the duodenal mouse tissue using

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EDTA, and confirmed by RT-PCR that Fgf10 is absent from the epithelial fraction as

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shown in figure 2D. Immunohistochemical analyses using antibodies against the

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receptors Fgfr1 and Fgfr2 (detecting both isoforms b and c) revealed that Fgfr1 (Figure

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2E-G) and Fgfr2 (Figure 2H-J) are expressed throughout the adult mouse small intestine

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(n=3). Negative controls with IgG and secondary antibodies alone did not show any

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staining (data not shown). Higher magnifications of the crypts containing the stem cell

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niche showed expression in the crypts of both Fgfr1 (Figure 2E’-G’) and Fgfr2 (Figure

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2H’-J’). Using RT-PCR, we showed that Fgf1, Fgf7 and Fgf10 were expressed in all

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three segments (duodenum, jejunum and ileum) of the adult mouse small intestine as

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shown in figure 2D, while Fgf3, Fgf20 and Fgf22 were not detected (Figure 2D).

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Importantly, Fgf10 seems to be expressed at higher levels in the duodenum as

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compared to the jejunum and ileum, which likely explains the lack of detection of LacZ

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activity in the jejunum and ileum of the Fgf10LacZ animals. Embryonic day E14.5 whole

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wild type embryo was used as a positive control for the various gene expressions.

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Furthermore, we did not detect any non-specific bands for any of the genes tested.

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Fgf10 overexpression increased crypt depth and villus height in the mouse small

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intestine

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In order to investigate the impact of Fgf10 signaling on the adult small intestine

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independently from development, we generated inducible gain- and loss-of-function

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mouse models. First, we crossed CMV-Cre animals with Rosa26-rtTA to generate an

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ubiquitous expression of the rtTA promoter. Then, we crossed these animals with a

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tet(O)Fgf10 promoter allowing overexpression of mouse Fgf10 under the conditional

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control of the Rosa26-rtTA promoter, hereafter referred to as Rosa26rtTA/+; tet(O)Fgf10/+.

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Fgf10 overexpression was achieved by feeding 4 weeks-old mice a doxycycline-

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containing diet for 10 days. Single transgenic littermates exposed to doxycycline (Control

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Dox) or double transgenic heterozygous mice on normal diet (Control no Dox) served as

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controls.

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To investigate the effects of Fgf signaling loss, we crossed the CMV-Cre; Rosa26-rtTA

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animals with mice harboring a tet(O)sFgfr2b promoter to generate mice overexpressing

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a soluble form of the mouse Fgfr2b receptor under the conditional control of the Rosa26-

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rtTA promoter similar to the Fgf10 overexpressing mice, hereafter referred to as

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Rosa26rtTA/+; tet(O)sFgfr2b/+. In these mice, exogenous sFgfr2b acts as a decoy receptor

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and upon exposure to doxycycline, it binds all Fgfr2b ligands available in the gut thus

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preventing their action. These mice were exposed to doxycycline food starting at 4

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weeks of age for a period of one month. This mouse model has been previously

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validated during embryonic development, where ubiquitous overexpression of sFgfr2b

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phenocopies Fgf10 null mice (7). Single transgenic animals treated with doxycycline or

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double transgenic animals not exposed to doxycycline were identical to wild type

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controls (31). The mice from both strains were monitored for body weight changes

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during the treatment period. The overexpression of Fgf10 for 10 days and the

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overexpression of sFgfr2b for one month or 3 months did not affect the body weight of

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the mutant mice compared to the controls (data not shown).

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Histological analyses were performed on the Rosa26rtTA/+; tet(O)Fgf10/+, Rosa26rtTA/+;

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tet(O)sFgfr2b/+ and the control littermates treated or not with doxycycline. Hematoxylin

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and eosin staining did not show any macroscopic differences in the gross histology of

369

the jejunum between Rosa26rtTA/+; tet(O)sFgfr2b/+ and controls (Figure 3A-C, 3A’-C’ and

370

3A’’-C’’), while an altered histology was observed in the Rosa26rtTA/+; tet(O)Fgf10/+ as

371

compared to the controls (Figure 3D,D’, D’’ vs A-B, A’-B’, A’’-B’’). There was no

372

difference in the control littermates between single transgenic on doxycycline food for 10

373

days or one month, or double transgenic animals on regular diet (B-B’’ vs A-A’’). The

374

mice overexpressing Fgf10 displayed longer villi and deeper crypts (Figure 3D-D’’). In

375

order to validate the animal models, we assessed the expression of Fgf10 by qRT-PCR

376

and showed a robust increase in Fgf10 expression in the animals harboring Rosa26rtTA

377

and tet(O)Fgf10 transgenes (Figure 3E). Fgf10 expression was very low in the jejunum

378

and ileum of controls treated or not with dox and undetectable in the mutants

379

overexpressing sFgfr2b. Moreover, after 10 days of doxycycline treatment, the mutant

380

mice developed a wet hair appearance and swelling of the eyelids and the tongue as

381

previously reported (38).

382 383

We also assessed the expression of the exogenous sFgfr2b transgene, and

384

demonstrated that it is highly expressed in all the animals harboring Rosa26rtTA and

16

385

sFgfr2b transgenes but not detectable in any of the other groups (Figure 3F). Both Fgf10

386

and sFgfr2b expression were similar in the controls treated or not with doxycycline.

387 388

We next measured the crypt depth (Figure 3G), villus height (Figure 3H) and the ratio

389

villus/ (villus + crypt) (Figure 3I) in the three segments of the intestine of the animals

390

overexpressing Fgf10, sFgfr2b and the controls. Crypt depth was significantly increased

391

in the duodenum, jejunum and ileum of the animals overexpressing Fgf10 but not in the

392

animals overexpressing sFgfr2b (Figure 3G). Villus height was significantly increased in

393

the duodenum, jejunum and ileum of the animals overexpressing Fgf10, with no change

394

in the animals overexpressing sFgfr2b as compared to littermate controls (Figure 3H).

395

The ratio villus/ (villus + crypt) was unchanged between the controls and the animals

396

overexpressing Fgf10 or sFgfr2b (Figure 3I; n=6; p=0.4 for duodenum; p=0.5 for jejunum

397

and p=0.8 for ileum). In addition, crypt fission was not affected in the mutants

398

overexpressing Fgf10 (Figure 3D’’) or sFgfr2b (Figure 3C’’) compared to controls treated

399

or not with dox (Figure 3A’’ and B’’) as shown in the high power field of crypts.

400 401

Fgf10 overexpression increases cell proliferation in the duodenum and decreases

402

cell death in the jejunum and ileum

403

FGF10 is known to promote cell proliferation during the development of the

404

gastrointestinal tract, as well as during gut adaptation (12, 33, 39, 41). Therefore, we

405

assessed the impact of Fgf10 signaling on intestinal epithelial cell proliferation. PCNA

406

staining (Figure 4A-D) on intestinal sections and quantification of the number of PCNA-

407

positive cells per crypt (Figure 4I) showed a significant increase in cell proliferation in the

408

duodenum of the mice overexpressing Fgf10 as compared to controls treated or not with

409

dox (Figure 4D,D’ vs A,A’ and B,B’ ; n=6, p=0.002), while no significant change in cell

410

proliferation was observed in the jejunum or ileum (Figure 4I; n=6; p=0.11 and 0.323

17

411

respectively). Similar results were obtained using another cell proliferation marker,

412

Phospho-Histone h3 (Figure 4E-H). Quantification of the Phh3-positive cells per crypt

413

showed a significant increase in the duodenum of the animals overexpressing Fgf10

414

(Figure 4J, n=6, p=0.035), but not in the jejunum or ileum (n=6, p=0.9 and 0.25

415

respectively). However, overexpression of sFgfr2b did not affect cell proliferation in any

416

of the intestinal segments studied as shown by PCNA staining in Figures 4C,C’ vs B,B’

417

and A,A’ and by Phh3 staining shown in Figures 4G vs 4E-F, and Figure 4J. Since

418

proliferation was only increased in the duodenum, we assessed cell death as a possible

419

explanation for the increase in crypt depth and villus height in the jejunum and ileum of

420

the Fgf10-overexpressing animals. We performed active (cleaved) caspase 3 IHC on

421

dox-treated controls and the animals overexpressing Fgf10, and counted the number of

422

active caspase-3 positive cells per 100 villi. We observed no change in cell death in the

423

duodenum of the animals overexpressing Fgf10 compared to controls, while there was a

424

significant decrease in cell death in the jejunum and ileum of these animals as compared

425

to controls (Figure 4K) (n=5, p=0.005 and 0.045 respectively for jejunum and ileum).

426 427

Fgf10 overexpression increases goblet cells and decreases Paneth cells in vivo

428

Next, we aimed to determine the effect of Fgf10 on epithelial cell differentiation in the

429

mouse

430

Chromogranin A (a marker of enteroendocrine cells) and lysozyme (a marker of Paneth

431

cells), as well as Alcian blue staining to assess goblet cell differentiation, on tissue

432

sections from mice overexpressing Fgf10, sFgfr2b and doxycycline treated controls.

433

There was no change in Chromogranin A staining in the jejunum between the animals

434

overexpressing Fgf10, sFgfr2b and the controls (Figure 5 C, B and A respectively). The

435

quantification of the cells stained for Chromogranin A did not show any difference in the

436

mice overexpressing Fgf10 or soluble Fgfr2b (Figure 5D). Lysozyme staining, used to

adult

small

intestine.

We

performed

immunofluorescent

staining

for

18

437

label Paneth cells, demonstrated decreased staining in the small intestinal segments of

438

the mice overexpressing Fgf10 (Figure 5G, jejunum shown) as compared to doxycycline-

439

treated controls (Figure 5E). Quantification of the number of Paneth cells in all three

440

segments of the mouse intestine (duodenum, jejunum and ileum) confirmed a significant

441

decrease in the number of Paneth cells in the jejunum and ileum of the mice

442

overexpressing Fgf10 (n=6, p=0.02 and 0.0008 respectively) but not in the duodenum

443

(p=0.099) (Figure 5H). In contrast, no change in the number of Paneth cells was

444

observed in the duodenum, jejunum or ileum of the animals overexpressing soluble

445

Fgfr2b (Figure 5F,H). Alcian blue staining showed increased goblet cells in the jejunum

446

of the mice overexpressing Fgf10 (Figure 5K) as compared to dox-treated controls

447

(Figure 5I). No change in Alcian blue staining was observed in the jejunum of the mice

448

overexpressing sFgfr2b (Figure 5J) as compared to dox-treated controls (Figure 5I).

449

Quantification of the percentage of goblet cells compared to the total number of epithelial

450

cells showed significant increase in the duodenum, jejunum and ileum (n=6; p=0.0003,

451

p=0.0005 and p=0.0002 respectively) in the mice overexpressing Fgf10 as compared to

452

dox-treated controls (Figure 5L). No change was observed in the mice overexpressing

453

sFgfr2b (Figure 5L). Taking into consideration the distribution of goblet cells along the

454

crypt/villus axis, we investigated whether there was an increase in goblet cells in both

455

crypt and villus compartments of the mice overexpressing Fgf10. The number of goblet

456

cells was significantly higher in the crypts (Figure 5M) of the mice overexpressing Fgf10

457

compared to dox-treated controls in the duodenum, jejunum and ileum (Figure 5M, n=6,

458

P=0.005, 0.002 and 0.01 respectively). In addition, the number of goblet cells was

459

significantly higher in the villi (Figure 5M) of the mice overexpressing Fgf10 compared to

460

dox-treated controls in the duodenum, jejunum and ileum (Figure 5M, n=6, P=0.001,

461

0.0001 and 0.008 respectively).

462 19

463

FGF10 induces goblet cell differentiation in intestinal enteroids

464

Since the overexpression of Fgf10 was achieved in a ubiquitous manner, we next asked

465

whether Fgf10 could act directly on the epithelium to control intestinal cell proliferation

466

and/or differentiation. For that purpose, we used an epithelial-only culture system (35),

467

where crypts from mouse intestine were embedded in Matrigel and grown in the

468

presence of a mixture of growth factors (EGF, Noggin, R-Spondin), with or without

469

recombinant human FGF10 (200 ng/mL). After 4 days in culture in the presence or

470

absence of rhFGF10, the cultures were fixed, immunostained for Phospho-histone h3 to

471

assess epithelial proliferation, and imaged as described in the methods section to obtain

472

a 3D re-construction of each crypt bud (Figure 6 A-B). In addition, we quantified of the

473

number of Phh3-positive cells normalized to enteroid volume. We did not observe any

474

difference in the proliferation in the presence or absence of FGF10 (Figure 6C).

475 476

To determine the effect of FGF10 on epithelial cell differentiation, we analyzed the gene

477

expression of Muc-2 and Lysozyme in the cultures (Figure 6E). In the presence of

478

rhFGF10, we found a significant increase in Muc-2 expression as compared to untreated

479

enteroids (n=6, p=0.01), reflecting an increase in goblet cells. In addition, there was a

480

significant decrease in Lysozyme in the FGF10-treated enteroids as compared to

481

untreated (n=6, p=0.014). In order to determine whether FGF10 affected the stem cell

482

niche, we analyzed the expression of several genes important for the differentiation and

483

commitment of the intestinal stem cells. There was no change in the expression of Atoh1

484

or Klf4 in the FGF10-treated crypts compared to controls suggesting that FGF10 does

485

not affect the global secretory progenitor pool. An increase in Spdef expression was

486

observed in the FGF10-treated enteroids (Figure 6D). Several stem cell markers were

487

decreased in the FGF10-treated enteroids as compared to control enteroids such as

488

Lgr5 (n=4, p=0.028), Hopx (n=5, p=0.0072), Lrig1 (n=5, 0.0032), Ascl2 (n=5, p=0.0033)

20

489

and Sox9 (n=4, p=0.05). Next we sought to determine whether this effect of FGF10 in

490

driving goblet cell differentiation is dependent on Notch signaling. Therefore, we

491

assessed the expression of Hes1 and found a significant decrease in Hes1 expression in

492

FGF10-treated vs untreated enteroids (n=4, p=0.009). It has been reported that Paneth

493

cell differentiation requires high Wnt activity, therefore we tested the effect of FGF10 on

494

Wnt activation using enteroids from the ileum of Axin2-LacZ reporter mice. We showed

495

that LacZ staining was significantly decreased in the presence of rhFGF10 (Figure 6D).

496

These data were confirmed by qRT-PCR, where FGF10 significantly decreased Axin2

497

expression (n=5, p=0.016).

498 499

Enteroids from the ileum of animals overexpressing Fgf10 were also cultured in the

500

presence of doxycycline to induce Fgf10 overexpression. Treating wild type enteroids

501

with doxycycline did not affect cell proliferation or expression of cell differentiation

502

markers (data not shown), consistent with previous studies (18). Immunofluorescence

503

staining of enteroid sections cultured for 3 days in absence or presence of 2 g of

504

doxycycline did not show any difference in cell proliferation as shown by Phh3 staining

505

(Figure 6G,H) and quantification (Figure 6H). We performed staining for Mmp7 (Paneth

506

cells), Muc2 (goblet cells) and Lysozyme (Paneth cells) to assess cell differentiation in

507

the

508

overexpression increased the number of Muc2 positive cells (Figure 6J vs I, 6K, n=4,

509

p=0.003). A decrease in the number of Mmp7 positive cells (Figure 6J vs I, 6K, n=4,

510

p=0.008) and Lysozyme positive cells (Figure 6M vs L, 6N, n=4, p= 0.01) was observed

511

as compared to controls. Interestingly, in the enteroids overexpressing Fgf10, the

512

number of cells positive for both Mmp7 and Muc-2 was increased by 3 fold as compared

513

to controls (Figure 6O, n=4, p=0.04), suggesting that following Fgf10 overexpression,

514

goblet cells replace Paneth cells in the enteroids. Moreover, following 3 days

enteroids

following

Fgf10

overexpression.

We

demonstrated

that

Fgf10

21

515

doxycycline administration in mice, we observed the presence of double positive cells for

516

Mmp7 and Muc2 in the ileum of animals overexpressing Fgf10 (Figure 6Q) compared to

517

dox-treated controls (Figure 6P). Fgf10 overexpression was confirmed by qRT-PCR, the

518

enteroids treated with dox had a 5-fold increase in Fgf10 expression (Figure 6R, n=4,

519

p=0.02). Moreover, Fgf10 overexpression significantly decreased the Lgr5 and Lrig1

520

expression in the enteroids (Figure 6S, n=4, p=0.02 and 0.008 respectively).

521

22

522

Discussion

523

This study describes the effect of Fgf10 overexpression on the adult mouse small

524

intestine. While Fgf10 and Fgf signaling in general have been extensively studied during

525

intestinal development, little is known their roles in adulthood. In addition, the expression

526

of FGFR1 and FGFR2, as well as FGF ligands in the human intestine, have not been

527

described. We demonstrated that both FGFR1 and FGFR2 are expressed in the human

528

ileum. Previous reports have shown that several human tissues express FGFR1 and

529

FGFR2 such as stomach, pancreas, salivary gland and duodenum (15). Both FGFR1

530

and FGFR2 are expressed throughout the epithelium and the mesenchyme of the ileum.

531

It was previously reported that FGF1 and FGF2 are widely expressed in normal adult

532

tissue- mainly in the kidney, skin and liver (16). Here we showed that FGF3 and FGF7

533

but not FGF1, 3 or 22 are expressed in human ileum. Moreover, FGFR1b and FGFR2b

534

(b isoforms), the receptors for FGF10, are both expressed in the human ileum. To our

535

knowledge, this is the first report of expression of FGF10 family members and FGF10

536

receptors in human ileum.

537 538

We have recently shown that Fgfr2b ligands: Fgf1, Fgf7, Fgf10 and Fgf22 are expressed

539

in adult mouse stomach (38). Here, we provide evidence that Fgf1, Fgf7 and Fgf10 are

540

expressed throughout the adult mouse intestine while in Fgf10LacZ reporter, beta-

541

galactosidase staining is detected only in the duodenum. This could be due to the lack of

542

sensitivity of the reporter insertion or to a lower expression of Fgf10 in the jejunum and

543

ileum as compared to the duodenum. Our data showed that FGFR1 and FGFR2 have

544

similar distribution in the human compared to the mouse ileum. However, Fgfr2b is

545

considered to be the main receptor of Fgf10 given the phenotypic similarities between

546

Fgf10-null and Fgfr2b-null mice (7, 25). The presence of FGF10 and its receptors,

547

FGFR1b and FGFR2b in human and mouse intestine, suggests an important role for

23

548

FGF10 signaling in intestinal homeostasis. Moreover, given the similar distribution of

549

FGFR1 and FGFR2 in human and mouse, we propose that FGF10/FGFR2b signaling

550

plays a similar and important role in small intestine both in mouse and human.

551 552

FGF10 is known as a mitogen during the development of several organs such as the

553

trachea (34) and colon (12, 33). It also promotes cell proliferation during homeostasis in

554

mouse mammary gland (32), incisors (31) and stomach (38). Our data suggest that

555

Fgf10 overexpression in the mouse adult small intestine increases crypt depth and villus

556

height in the duodenum, likely as a result of increased proliferation. In the jejunum and

557

ileum, there was no significant difference in proliferation. However, crypt depth and villus

558

heights were increased in both segments upon Fgf10 overexpression. This is likely due

559

a decrease in cell turnover in the intestine as a result of decreased cell death shown by

560

decreased active caspase-3 positive cells in the jejunum and ileum following Fgf10

561

overexpression. These results are consistent with our previous findings that Fgf10

562

enhances epithelial cell survival during colon development (33). Other studies have

563

shown that Fgf10 does not affect cell proliferation in embryonic intestinal development

564

including in the duodenum (29). This discrepancy between our studies could be due to

565

the fact that we employed a ubiquitous overexpression system and analyzed adult

566

organs while Nyeng et al used a Pdx1 driver and studied organogenesis. Similar to our

567

results, a recent study showed that FGF7 increases villus height and crypt depth.

568

However, that was accompanied with increased proliferation in the jejunum of adult mice

569

(5).

570 571

Fgf10 is known to play an important role in epithelial cell differentiation during

572

organogenesis in several organ systems (2, 34, 39, 46), including the intestine (29).

573

Nyeng et al showed that overexpressing Fgf10 using a Pdx1 driver resulted in a

24

574

decrease in Paneth cells, corroborating our results in adult mouse intestine. During

575

embryonic development, overexpression of Fgf10 did not seem to affect goblet cell

576

differentiation (29), while here we provide evidence that overexpressing Fgf10 in adult

577

mouse intestine increases goblet cell differentiation in both crypt and villus and

578

decreases Paneth cell numbers. In a colon epithelial cell line, Iwakiri et al showed that

579

FGF7 regulates the goblet cell silencer inhibitor and induces the differentiation of goblet

580

cells in vitro (17). While we show that activating Fgf10 signaling reduces the numbers of

581

Paneth cells, other Fgf receptor activation such as via Fgfr3 acts in an opposite manner.

582

Lack of Fgfr3 results in fewer Paneth cells in the mouse intestine (47). In contrast, Fgfr3

583

activation was shown to induce Paneth cell differentiation, either through MAPK or beta-

584

catenin activation (3). Therefore, it seems as though Fgf10 and Fgfr3 have opposing

585

signaling activities in the small intestine.

586 587

Our data raise interesting questions about regulation of the dynamics of the goblet and

588

Paneth cell populations in the intestine. The increase in goblet cells in the crypt along

589

with decreased Paneth cells may reflect goblet cells arising from mature Paneth cells in

590

the crypt compartment. This possibility is consistent with our observation of “intermediate”

591

cells expressing both Paneth and goblet cell markers at 3 days following FGF10

592

induction in either enteroid culture or mice (Figure 6), which are not observed in vivo 10

593

days after induction (data not shown). The possibility of this transitional cell type has

594

previously been demonstrated, for example, in the case of altered Notch signaling (45).

595

Alternatively, it may be that goblet cell differentiation is accelerating in concert with

596

extrusion or death of Paneth cells. Further investigation will be needed to distinguish

597

between these possibilities.

598

25

599

It is still unclear what controls the balance between goblet and Paneth cell lineage

600

specification, though it is well established that Paneth cell formation is dependent on

601

Wnt/beta-catenin/TCF4 signaling (43). In our experiments, FGF10 decreased transcript

602

levels of the Axin2 gene as well as LacZ expression in enteroids from Axin2-LacZ

603

reporter mice, indicating that FGF10 inhibits Wnt signaling in these enteroids (Figure 6).

604

The inhibition of Wnt signaling was also accompanied by a decrease in the expression of

605

a subset of stem cell-associated wnt target genes such as Lgr5, Lrig1, Ascl2 and Hopx.

606

Furthermore, FGF10 decreased Hes1 expression. These results are in accordance with

607

published data that Notch inhibition induces goblet cell hyperplasia (45), as in many

608

models ablation of Paneth cells results in a decrease of Lgr5-positive cells (35).

609 610

In summary, our studies implicate FGF signaling as an important regulator of the

611

balance between the different intestinal secretory cell types. Future studies in this area

612

are needed to elucidate how FGF10 affects these secretory lineages, and what signaling

613

mechanisms could be involved to compensate for the loss of the different stem cell

614

markers studied here. Understanding the role of FGF signaling in this context could lead

615

to therapeutically relevant insights for disease states associated with goblet cell

616

hyperplasia.

26

617

Grants

618

DAA acknowledges support from the Saban Research Institute and NIH/NCATS Grant #

619

UL1TR000130 via USC-CTSI. SD is supported by a training grant from NIH/

620

5T90DE021982-03. MRF is supported by NIH/NIDDK R01DK095004, a Senior

621

Research Award from the Crohn's and Colitis Foundation of America, an American

622

Cancer Society Research Scholar Award and a Research Career Development Award

623

from the Saban Research Institute. TG receives support from the California Institute for

624

Regenerative Medicine (CIRM) RN 2 00946-1 and RN3-06425. HRF and SB

625

acknowledge the support of NIH/NIDDK R01HD052609 and R01AI014032 (HRF).

626 627 628 629 630 631 632 633

27

634

References

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14. Hokuto I, Perl AK, and Whitsett JA. Prenatal, but not postnatal, inhibition of fibroblast growth factor receptor signaling causes emphysema. The Journal of biological chemistry 278: 415-421, 2003. 15. Hughes SE. Differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal human adult tissues. J Histochem Cytochem 45: 1005-1019, 1997. 16. Hughes SE, and Hall PA. Immunolocalization of fibroblast growth factor receptor 1 and its ligands in human tissues. Lab Invest 69: 173-182, 1993. 17. Iwakiri D, and Podolsky DK. Keratinocyte growth factor promotes goblet cell differentiation through regulation of goblet cell silencer inhibitor. Gastroenterology 120: 1372-1380, 2001. 18. Jarde T, Evans RJ, McQuillan KL, Parry L, Feng GJ, Alvares B, Clarke AR, and Dale TC. In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-ODeltaN89beta-catenin system. Oncogene 32: 883-893, 2013. 19. Jenny M, Uhl C, Roche C, Duluc I, Guillermin V, Guillemot F, Jensen J, Kedinger M, and Gradwohl G. Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium. EMBO J 21: 6338-6347, 2002. 20. Jensen J, Pedersen EE, Galante P, Hald J, Heller RS, Ishibashi M, Kageyama R, Guillemot F, Serup P, and Madsen OD. Control of endodermal endocrine development by Hes-1. Nat Genet 24: 36-44, 2000. 21. Kanard RC, Fairbanks TJ, De Langhe SP, Sala FG, Del Moral PM, Lopez CA, Warburton D, Anderson KD, Bellusci S, and Burns RC. Fibroblast growth factor-10 serves a regulatory role in duodenal development. J Pediatr Surg 40: 313-316, 2005. 22. Katz JP, Perreault N, Goldstein BG, Lee CS, Labosky PA, Yang VW, and Kaestner KH. The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development 129: 2619-2628, 2002. 23. Kelly RG, Brown NA, and Buckingham ME. The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev Cell 1: 435-440, 2001. 24. Mailleux AA, Kelly R, Veltmaat JM, De Langhe SP, Zaffran S, Thiery JP, and Bellusci S. Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage. Development 132: 2157-2166, 2005. 25. Mailleux AA, Spencer-Dene B, Dillon C, Ndiaye D, Savona-Baron C, Itoh N, Kato S, Dickson C, Thiery JP, and Bellusci S. Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development 129: 53-60, 2002. 26. Mellitzer G, Beucher A, Lobstein V, Michel P, Robine S, Kedinger M, and Gradwohl G. Loss of enteroendocrine cells in mice alters lipid absorption and glucose homeostasis and impairs postnatal survival. J Clin Invest 120: 1708-1721, 2010. 27. Mori-Akiyama Y, van den Born M, van Es JH, Hamilton SR, Adams HP, Zhang J, Clevers H, and de Crombrugghe B. SOX9 is required for the differentiation of paneth cells in the intestinal epithelium. Gastroenterology 133: 539-546, 2007. 28. Noah TK, Kazanjian A, Whitsett J, and Shroyer NF. SAM pointed domain ETS factor (SPDEF) regulates terminal differentiation and maturation of intestinal goblet cells. Exp Cell Res 316: 452-465, 2010. 29. Nyeng P, Bjerke MA, Norgaard GA, Qu X, Kobberup S, and Jensen J. Fibroblast growth factor 10 represses premature cell differentiation during establishment of the intestinal progenitor niche. Dev Biol 349: 20-34, 2011. 30. Parsa S, Kuremoto K, Seidel K, Tabatabai R, Mackenzie B, Yamaza T, Akiyama K, Branch J, Koh CJ, Al Alam D, Klein OD, and Bellusci S. Signaling by

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FGFR2b controls the regenerative capacity of adult mouse incisors. Development 137: 3743-3752. 31. Parsa S, Kuremoto K, Seidel K, Tabatabai R, Mackenzie B, Yamaza T, Akiyama K, Branch J, Koh CJ, Al Alam D, Klein OD, and Bellusci S. Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors. Development 137: 3743-3752, 2010. 32. Parsa S, Ramasamy SK, De Langhe S, Gupte VV, Haigh JJ, Medina D, and Bellusci S. Terminal end bud maintenance in mammary gland is dependent upon FGFR2b signaling. Dev Biol 317: 121-131, 2008. 33. Sala FG, Curtis JL, Veltmaat JM, Del Moral PM, Le LT, Fairbanks TJ, Warburton D, Ford H, Wang K, Burns RC, and Bellusci S. Fibroblast growth factor 10 is required for survival and proliferation but not differentiation of intestinal epithelial progenitor cells during murine colon development. Dev Biol 299: 373-385, 2006. 34. Sala FG, Del Moral PM, Tiozzo C, Alam DA, Warburton D, Grikscheit T, Veltmaat JM, and Bellusci S. FGF10 controls the patterning of the tracheal cartilage rings via Shh. Development 138: 273-282, 2011. 35. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, and Clevers H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469: 415-418, 2011. 36. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, and Cardona A. Fiji: an open-source platform for biologicalimage analysis. Nat Methods 9: 676-682, 2012. 37. Shroyer NF, Helmrath MA, Wang VY, Antalffy B, Henning SJ, and Zoghbi HY. Intestine-specific ablation of mouse atonal homolog 1 (Math1) reveals a role in cellular homeostasis. Gastroenterology 132: 2478-2488, 2007. 38. Speer AL, Al Alam D, Sala FG, Ford HR, Bellusci S, and Grikscheit TC. Fibroblast growth factor 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis. PLoS One 7: e49127, 2012. 39. Spencer-Dene B, Sala FG, Bellusci S, Gschmeissner S, Stamp G, and Dickson C. Stomach development is dependent on fibroblast growth factor 10/fibroblast growth factor receptor 2b-mediated signaling. Gastroenterology 130: 1233-1244, 2006. 40. Suzuki K, Fukui H, Kayahara T, Sawada M, Seno H, Hiai H, Kageyama R, Okano H, and Chiba T. Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine. Biochem Biophys Res Commun 328: 348-352, 2005. 41. Tai CC, Curtis JL, Sala FG, Del Moral PM, Chokshi N, Kanard RJ, Al Alam D, Wang J, Burns RC, Ford HR, Grishin A, Wang KS, and Bellusci S. Induction of fibroblast growth factor 10 (FGF10) in the ileal crypt epithelium after massive small bowel resection suggests a role for FGF10 in gut adaptation. Dev Dyn 238: 294-301, 2009. 42. Torashima Y, Levin DE, Barthel ER, Speer AL, Sala FG, Hou X, and Grikscheit TC. Fgf10 overexpression enhances the formation of tissue-engineered small intestine. J Tissue Eng Regen Med 2013. 43. van Es JH, Jay P, Gregorieff A, van Gijn ME, Jonkheer S, Hatzis P, Thiele A, van den Born M, Begthel H, Brabletz T, Taketo MM, and Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nat Cell Biol 7: 381-386, 2005. 44. van Es JH, van Gijn ME, Riccio O, van den Born M, Vooijs M, Begthel H, Cozijnsen M, Robine S, Winton DJ, Radtke F, and Clevers H. Notch/gammasecretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435: 959-963, 2005.

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45. VanDussen KL, Carulli AJ, Keeley TM, Patel SR, Puthoff BJ, Magness ST, Tran IT, Maillard I, Siebel C, Kolterud A, Grosse AS, Gumucio DL, Ernst SA, Tsai YH, Dempsey PJ, and Samuelson LC. Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. Development 139: 488-497, 2012. 46. Veltmaat JM, Relaix F, Le LT, Kratochwil K, Sala FG, van Veelen W, Rice R, Spencer-Dene B, Mailleux AA, Rice DP, Thiery JP, and Bellusci S. Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes. Development 133: 2325-2335, 2006. 47. Vidrich A, Buzan JM, Brodrick B, Ilo C, Bradley L, Fendig KS, Sturgill T, and Cohn SM. Fibroblast growth factor receptor-3 regulates Paneth cell lineage allocation and accrual of epithelial stem cells during murine intestinal development. American journal of physiology Gastrointestinal and liver physiology 297: G168-178, 2009. 48. Yang Q, Bermingham NA, Finegold MJ, and Zoghbi HY. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science 294: 21552158, 2001.

801 802 803

31

804

Figure legends

805 806

Figure 1: Expression of FGFR1, FGFR2 and their ligands in human ileum. (A-B)

807

H&E staining of normal human ileum showing crypt and villus structures (A) and higher

808

magnification of A (B). (C-D) IHC using anti-FGFR1 antibody from Santa Cruz (Flg) (C)

809

and anti-FGFR1 from Abcam (D) on human ileum. (E) Negative control using matching

810

anti-IgG. (E’) Negative control using the competition peptide for Flg antibody. (F-G) IHC

811

using anti-FGFR2 antibody from Santa Cruz (Bek) (F) and anti-FGFR2 antibody from

812

Abcam (G) on human ileum. (H) Negative control using matching anti-IgG. (H’) Negative

813

control using the competition peptide for Bek antibody. (I-J) qRT-PCR for FGFs ligands

814

(I, n=5 at least), FGFR1b and FGFR2b (J, n=9) on human ileum. Scale bars are 100 m.

815 816

Figure 2: Expression of FGFR1, FGFR2 and FGFR2b ligands in the small intestine.

817

(A-C) LacZ (blue) and nuclear red (pink) staining on tissue sections from duodenum (A),

818

jejunum (B) and ileum (C) of adult Fgf10-LacZ mice. (D) RT-PCR for Fgf10 and ß-actin

819

on separated epithelial and mesenchymal fractions from adult mouse duodenum (n=3).

820

(E-G) IHC staining for Fgfr1 on mouse adult duodenum (E), jejunum (F) and ileum (G).

821

(E’-G”) Higher magnifications of crypts from panels E, F and G. (H-J) IHC staining for

822

Fgfr1 on mouse adult duodenum (H), jejunum (I) and ileum (J). (H’-J’) Higher

823

magnifications of crypts from panels H, I and J. (K) qRT-PCR for Fgf10 family members

824

on adult mouse duodenum, ileum and jejunum and E14.5 whole mouse embryo (positive

825

control). Negative controls did not show any bands; n=3. In order to show the entire

826

villus height, the duodenum sections are shown at lower magnifications than the jejunum

827

and ileum panels. Scale bars are 100 m in A-C, F-G, I-J; 200 m in E-H and 20 m in

828

E’-G’ and H’-J’.

32

829 830

Figure 3: Increased villus height in the duodenum and jejunum after Fgf10

831

overexpression. (A-D) Hematoxylin and eosin staining of jejunum sections of control

832

not treated with doxycycline (A, A’, A’’), dox-treated controls (B, B’, B’’), Rosa26rtTA/-;

833

tet(o)sFgfr2b/+ (C, C’, C’’)) and Rosa26rtTA/-; tet(o)Fgf10/+ animals (D, D’, D’’). A’-A’’, B’-

834

B’’, C’-C’’, D’-D’’ are higher magnifications of A, B, C and D respectively; (E) qRT-PCR

835

validating the expression of Fgf10 in the controls vs Rosa26rtTA/-; tet(o)Fgf10/+ animals

836

(n=6) (E) qRT-PCR showing the expression of exogenous sFgfr2b transcripts in the

837

controls vs Rosa26rtTA/-; tet(o)sFgfr2b/+ animals (n=6) (G) Quantification of the crypt

838

depths (H) Quantification of the villus height (I) Quantification of the ratio villus/(villus +

839

Crypt). N=6 for each group, *p0.05. Scale bars are 500 m in A-D, 200 m in A’-D’ and

840

20 m in A’’-D’’.

841 842

Figure 4: Increased cell proliferation in the duodenum of mutants overexpressing

843

Fgf10. (A-D) IHC for PCNA on duodenum sections from controls not treated with dox (A),

844

dox-treated controls (B), Rosa26rtTA/-; tet(o)sFgfr2b/+ (C) and Rosa26rtTA/-; tet(o)Fgf10/+

845

animals (D). A’, B’, C’ and D’ are higher magnifications of the insets in A, B,C and D

846

respectively. (E-H) IF staining for Phh3 on duodenum sections from controls not treated

847

with dox (E), dox-treated controls (F), Rosa26rtTA/-; tet(o)sFgfr2b/+ (G) and Rosa26rtTA/-;

848

tet(o)Fgf10/+ animals (H). (I) Quantification of cell proliferation in the crypts as the

849

percentage of the number of PCNA+ positive to the number of total cells per crypt. (J)

850

Quantification of the number of Phh3+ cells per 100 crypts cell. (K) Quantification of cell

851

death in the villi as the number of active caspase 3 + cells per 100 villi. The results are

852

expressed as mean  SEM of at least 5 independent animals per group. *p0.05;

853

**p0.01. Scale bars are 200 m in A-D, 20 m in A’-D’ and E-H.

33

854 855

Figure 5: Fgf10 overexpression results in increased goblet cells and decreased

856

Paneth cells. (A-C) Chromogranin A IF staining in the jejunum of dox-treated control (A),

857

Rosa26rtTA/-; tet(o)sFgfr2b/+ (B) and Rosa26rtTA/-; tet(o)Fgf10/+ animals (C). (D)

858

Quantification of the number of Chromogranin A positive cells per total number of

859

epithelial cells. (E-G) Lysozyme staining (red) a marker of Paneth cells in the jejunum of

860

dox-treated control (E), Rosa26rtTA/-; tet(o)sFgfr2b/+ (F) and Rosa26rtTA/-; tet(o)Fgf10/+

861

(G). (H) Quantification of the number of Lysozyme positive cells per total number of

862

epithelial cells per crypt. (I-K) Alcian blue staining to label goblet cells in the jejunum of

863

dox-treated control (I), Rosa26rtTA/-; tet(o)sFgfr2b/+ (J) and Rosa26rtTA/-; tet(o)Fgf10/+

864

animals (K). (L) Quantification of the number of goblet cells per total number of epithelial

865

cells. (M) Quantification of the number of goblet cells per total number of epithelial cells

866

in the crypts of control Dox and Rosa26rtTA/-; tet(o)Fgf10/+. (N) Quantification of the

867

number of goblet cells per total number of epithelial cells in the villi of dox-treated control

868

and Rosa26rtTA/-; tet(o)Fgf10/+. The results in D, H, L, M and N are expressed as mean 

869

SEM of 6 independent animals per group. *p0.05; **p0.01; ***p0.001. Scale bars are

870

100 m.

871 872

Figure 6: FGF10 acts directly on the epithelium to induce goblet cells and

873

decrease Paneth cells and stem cell markers. (A-B) 3D images of whole mount IF

874

staining of Phh3 and Hoechst on enteroid cultures in absence (A) or presence of rh

875

FGF10 (200 ng/mL). (C) Quantification of the number of Phh3-positive cells per mm3.

876

Results are expressed as mean  SEM of 5 independent experiments. 3 enteroids were

877

analyzed in each experiment. (D) LacZ staining of ileal enteroids from Axin2-LacZ

878

reporter mouse in absence (control) or presence (+FGF10) of rh FGF10 and qRT-PCR

34

879

for Axin2 transcripts in WT enteroids treated or not with rh FGF10. (E) qRT-PCR on RNA

880

from wild type enteroids untreated (black bars) or treated (Gray bars) with 200 ng/mL of

881

rhFGF10 for Muc-2, Lysozyme, Atoh1, Klf4, Spdef, Ascl2, Hes1, Hopx, Lgr5, Lrig1 and

882

Sox9. (F-G) IF staining for Phh3 (red) and ECadherin (green) on enteroids isolated from

883

Rosa26rtTA/-; tet(o)Fgf10/+ treated (G) or not (F) with dox. (H) Quantification of the

884

percent of Phh3+ cells per total number of DAPI. (I-J) IF staining for Mmp7 (green) and

885

Muc2 (red) on enteroids isolated from Rosa26rtTA/-; tet(O)Fgf10/+ treated (J) or not (I)

886

with dox. (K) Quantification of the percent of Muc2+ and Mmp7+ cells per total number

887

of DAPI. (L-M) IF staining for Lysozyme (red) on enteroids isolated from Rosa26rtTA/-;

888

tet(o)Fgf10/+ treated (M) or not (L) with dox. (N) Quantification of the percent of

889

Lysozyme+ cells per total number of DAPI. (O) Quantification of the percent of double

890

positive cells for Mmp7 and Muc2 compared to the total number of Mmp7+ cells. (P-Q)

891

Mmp7 and Muc2 IF staining on ileum from control animals (P) and Rosa26-RtTA,

892

tet(o)Fgf10 animals treated with doxycycline for 3 days. Arrows show double positive

893

cells. (R) qRT-PCR confirming Fgf10 overexpression in the enteroids treated with dox

894

compared to the untreated enteroids. (S) qRT-PCR to assess gene expression of Lgr5

895

and Lrig1 following Fgf10 overexpression. Scale bars are 100 m in A-B, 50 m in F-G

896

and 25 m in I-J, L-M. * p0.05.

897 898

35

Gene

Probe

Ascl2

17

Atoh1

69

Axin2

50

Fgf10

80

s Fgfr2b

108

Hes1

20

Hopx

68

Klf4

62

Lgr5

60

Lrig1

89

Lysozyme

46

Muc2

76

Sox9

75

Spdef

22

Primer Orientation Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left

Sequence (5’-3’) AGGTCCACCAGGAGTCACC GAGAGCTAAGCCCGATGGA CTCTTCTGCAAGGTCTGATTTTT TGCGATCTCCGAGTGAGAG TGCCAGTTTCTTTGGCTCTT CTGCTGGTCAGGCAGGAG AACAACTCCGATTTCCACTGA CGGGACCAAGAATGAAGACT GAAGGAGATCACGGCTTCC AGACAGATGATACTTCTGGGACTGT CCATGATAGGCTTTGATGACTTT TGCCAGCTGATATAATGGAGAA GCGCTGCTTAAACCATTTCT ACCACGCTGTGCCTCATC GAGTTCCTCACGCCAACG CGGGAAGGGAGAAGACACT CAGCCAGCTACCAAATAGGTG CTTCACTCGGTGCAGTGCT CAGGTGGCTGTCACAAAGG TGCAAAGATGAAGAACCTTAAAGA CACCACCCTCTTTGCACATT TGGGATCAATTGCAGTGCT CGTGTCATATTTGCACCTCTTG GTCAGAGACGCCCTTTGC TCCACGAAGGGTCTCTTCTC CAGCAAGACTCTGGGCAAG GACGAGTCCACCTCACCATC CCCACCTGGACATCTGGA

    Table 1. Primer sequences and probe numbers used in qRT-PCR analyses of mouse gene expression