Examining the Role of Paneth Cells in the Small Intestine byLineage Ablation in Transgenic Mice*

(Received for publication, February 12, 1997, and in revised form, May 1, 1997)

Emily M. Garabedian, Lisa J. J. Roberts, M. Shane McNevin, and Jeffrey I. Gordon‡

From the Department of Molecular Biology and Pharmacology, Washington University School of Medicine,St. Louis, Missouri 63110

The Paneth cell lineage is one of four epithelial lin-eages derived from the adult mouse small intestine’smultipotent stem cell. Mature Paneth cells secrete anti-microbial peptides (cryptdins), growth factors, as wellas two gene products, a secreted phospholipase A2 andmatrilysin, that has been implicated as modifiers of ad-enoma formation in mice containing a mutation in thetumor suppressor Apc. Immature Paneth cells are lo-cated just above and below the cell layer, in intestinalcrypts, that has been proposed to contain the multipo-tent stem cell. Paneth cells differentiate during a down-ward migration to the crypt base. The location and di-rection of Paneth cell migration, their high density andlong residency time at the crypt base, and the nature oftheir secreted gene products, suggest that they may in-fluence the structure and/or function of the stem cellniche. Paneth cell ablation can therefore be viewed asan experimental manipulation of the cellular microen-vironment that purportedly contains the stem cell andits immediate descendants. Two types of ablation exper-iments were performed in transgenic mice. Nucleotides26500 to 134 of the mouse cryptdin-2 gene (CR2) wereused to express an attenuated diphtheria toxin A frag-ment. Light and electron microscopic immunohisto-chemical analyses of several pedigrees of postnatal day28 to 180 animals established that ablation of Panethcells is accompanied by an increase in the proportion ofundifferentiated crypt base columnar cells. These cellsnormally co-exist with Paneth cells. The ablation doesnot produce a detectable effect on the proliferation orterminal differentiation programs of the other three lin-eages or on host-microbial interactions. The last conclu-sion is based on the ability of crypts to remain free ofmicrobes detectable by Gram and Warthin-Starry stainsand by retention of the normal crypt-villus distributionof components of the diffuse gut-associated lymphoidtissue. CR2-directed expression of simian virus 40 largeT antigen also results in a loss of mature Paneth cellsbut produces a marked amplification of crypt cells hav-ing a morphology intermediate between Paneth andgranule goblet cells. EM immunohistochemical analysessuggest that intermediate cells can differentiate to ma-ture goblet cells but not to Paneth cells, as they migrateup the crypt-villus axis. Our findings suggest that (i)stemness in the crypt is not defined by instructive inter-actions involving the Paneth cell; (ii) expressing a Pan-

eth cell fate may require that precursors migrate to thecrypt base; (iii) antimicrobial factors produced by Pan-eth cells are not required to prevent colonization ofsmall intestinal crypts; and (iv) this lineage does notfunction to maintain the asymmetric crypt-villus distri-bution of components of the diffuse gut-associatedlymphoid tissue.

The structural and functional organization of the adultmouse small intestinal epithelium lends itself to studying boththe regulation and integration of cellular proliferation, differ-entiation, and death programs. The epithelium contains fourprincipal cell types: absorptive enterocytes (comprising .80%of the total population), enteroendocrine cells, mucus-produc-ing goblet cells, and Paneth cells. All four lineages are derivedfrom a multipotent stem cell that is functionally anchored nearthe base of each of the small intestine’s 1.1 million crypts ofLieberkuhn (1–4). Cell division is confined to these crypts (5).Enterocytes, enteroendocrine, and goblet cells migrate out ofthe crypt and up an adjacent villus. Migration is highly orderedand associated with terminal differentiation. Cell death occursnear the villus tip where cells are exfoliated into the lumen (6,7). Proliferation, differentiation, and death take place in aspatially well-organized continuum that extends from the cryptto the apex of a villus. This sequence is completed rapidly (2–5days in the case of enterocytes, enteroendocrine, and gobletcells; Refs. 1 and 8–10) and is recapitulated throughout thelifespan of the mouse.

The Paneth cell lineage differs from the others in a numberof notable ways. It is the only lineage that executes its terminaldifferentiation program during a downward migration from thestem cell zone to the crypt base (11). It is the longest livedlineage, and the only one that exists entirely within the prolif-erative compartment. Each crypt contains 30–50 mature Pan-eth cells that survive for 18–23 days before degenerating andundergoing phagocytosis by their neighbors (11–13). Panethcell age correlates with position in the crypt; the most maturecells are located at or near the crypt base (2). The size of thePaneth cell’s apical secretory granules also correlates with age;larger granules are produced by older cells (2, 11).

The function of the Paneth cell has not yet been clearlydefined. Residency at the crypt base places this lineage in aposition to release products from its apical granules that couldaffect establishment and/or maintenance of the stem cell’sniche or influence the properties of the stem cell’s descendants.A number of factors exported by Paneth cells could regulateepithelial proliferation and differentiation programs. They in-clude tumor necrosis factor-a (14), guanylin (15), and epider-mal growth factor (16). Two Paneth cell products have beenimplicated as modifiers of adenoma formation in mice heterozy-gous for a mutation in the adenomatous polyposis coli gene,ApcMin (17). Production of matrilysin, a matrix metalloprotein-

* This work was supported in part by National Institutes of HealthGrant DK 37960 and by a grant from Amgen. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed: Dept. of MolecularBiology and Pharmacology, Box 8103, Washington University School ofMedicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243; Fax: 314-362-7047; E-mail: jgordon@pharmdec.wustl.edu.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 38, Issue of September 19, pp. 23729–23740, 1997© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 23729

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

ase, is limited to the Paneth cell lineage in the adult mouseintestine (18). The protein is expressed in a high percentage ofearly stage human colorectal neoplasms and Min adenomas(19). Min/1 mice homozygous for a null allele of the matrilysingene have 60% fewer adenomas than animals with the wildtype allele, suggesting that the enzyme functions as suppressorof tumorigenesis (19). Pla2 g2a encodes a phospholipase A2

that is secreted from Paneth cells (20–22). This gene is a strongcandidate for Mom1, a semi-dominant modifier of adenoma sizeand multiplicity in Min/1 animals (23–25).

Paneth cells also export lysozyme (26, 27) and a family ofdefensin-related anti-microbial peptides known as cryptdins(28). The intestine contains a complex microflora. Componentsof this microflora are able to establish stable niches at partic-ular positions along the duodenal-ileal axis (29). The fact thatdifferent cryptdins exhibit distinct developmental and spatialpatterns of expression along this axis (30) suggests that Panethcells could play a role in modulating the composition of themicrobiota or contribute to mucosal barrier functions.

We have examined the contribution of this lineage to epithe-lial and microbial homeostasis by generating two types oftransgenic mice in which mature Paneth cells have beeneliminated.

EXPERIMENTAL PROCEDURES

Construction of Transgenes—A 2.7-kb1 DNA fragment containingsimian virus 40 large T antigen (SV40 TAg) was excised from pIF-TAg-hGH (31) with BamHI and subcloned into the BamHI site of pCR-H1(32). This yielded pCR2-TAg which contained SV40 TAg under thecontrol of nucleotides 26500 to 134 of the mouse cryptdin-2 gene (33).

Complete digestion of pIF-TAg-hGH with BamHI, followed by partialdigestion with EcoRI, allowed purification of a DNA fragment contain-ing pBluescript SK1 (Stratagene) with nucleotides 13 to 12150 of thehuman growth hormone gene (hGH; Ref. 34). This fragment and aBamHI/EcoRI fragment containing cryptdin-226500 to 134 and SV40 TAgfrom pCR2-TAg were ligated together, producing pCR2-TAg-hGH.pCR2-TAg-hGH was subsequently cut with BamHI, treated with Kle-now, and ligated to a 0.6-kb HincII fragment containing an attenuateddiphtheria toxin A fragment (tox176; Ref. 35). The resulting plasmid,pCR2-tox176-hGH, contained tox176 immediately downstream of crypt-din-226500 to 134 and immediately upstream of hGH13 to 12150. tox176was placed in exon1 of hGH to enhance the chances of efficientlyexpressing the toxin. hGH will not be produced from the RNA transcriptof cryptdin-226500 to 134-tox176-hGH13 to 12150: the initiator Met and thefirst stop codon are from the open reading frame of tox176, and there is noribosomal re-entry site to re-initiate translation at the downstream initi-ator ATG of hGH.

Generation of Transgenic Mice—A 9.2-kb fragment containing crypt-din-226500 to 134 and SV40 TAg (CR2-TAg) was released from pCR2-TAgby digestion with NotI and EcoRI. pCR2-tox176-hGH was digested withNotI and XhoI to liberate a 9.4-kb DNA fragment containing cryptdin-226500 to 134-tox176-hGH (CR2-tox176). An 8.3-kb fragment containingcryptdin-226500 to 134 linked to hGH13 to 12150 (CR2-hGH) was releasedfrom pCR-H1 with EcoRI (32). Each fragment was purified by agarosegel electrophoresis followed by glass bead extraction (Geneclean, Bio101) and used for pronuclear injection of FVB/N oocytes. Oocytes weresubsequently transferred to pseudopregnant Swiss Webster femalesusing standard techniques (36).

Live born mice were screened for the presence of transgenes byextracting tail DNA and performing polymerase chain reactions usingprimers that anneal to hGH DNA (CR2-tox176; CR2-hGH; 59-AGGTG-GCCTTTGACACCTACCAGG-39 and 59-TCTGTTGTGTTTCCTCCCT-

GTTGG-39) or to SV40 TAg DNA (CR2-TAg; 59-ATGAATGGGAGCAGT-GGTG-39 and 59-GCAGACACTCTATGCCTGTGTGG-39). Thepolymerase chain reaction mixtures (final volume 5 25 ml) contained 50mM KCl, 20 mM Tris, pH 8.4, 2 mM MgCl2, 200 mM dNTPs, primers (1 mM

each), 0.7 unit of Taq DNA polymerase (Boehringer Mannheim), andapproximately 0.5 mg of genomic DNA. The following cycling conditionswere used to amplify an hGH fragment from CR2-tox176 and CR2-hGHDNAs: denaturation, 1 min at 94 °C; annealing, 1.5 min at 55 °C; andextension, 2 min at 72 °C for 30 cycles. For CR2-TAg, denaturation wasperformed at 95 °C and annealing at 58 °C.

Four CR2-hGH founders were identified from 38 live born mice, 2CR2-tox176 founders from 67 mice, and 10 CR2-TAg founders from 87animals. Pedigrees were established from each of the CR2-hGH andCR2-tox176 founders and from 8 of the CR-TAg founders. All pedigreeswere maintained by crosses to normal FVB/N littermates. Pedigree 61,containing CR2-hGH, has been described in an earlier publication (32).

Maintenance of Animals—Mice were housed in microisolator cagesunder a strictly controlled light cycle (lights on at 0600 h and off at1800 h) and given a standard irradiated chow diet ad libitum (Picorodent chow 20, Purina Mills). Routine screens for hepatitis, minute,lymphocytic choriomeningitis, ectromelia, polyoma, sendai, pneumonia,and MAD viruses, enteric bacterial pathogens, and parasites werenegative. Specific pathogen-free transgenic animals and their nontrans-genic littermates were sacrificed between postnatal days 28 (P28) andP180.

Histochemical Stains—Immediately after sacrifice, the small intes-tine was removed en bloc, flushed with ice-cold phosphate-bufferedsaline (PBS), fixed in 10% buffered formalin (Fisher) for 4–6 h, andthen washed in 70% ethanol overnight at room temperature. The intes-tine was embedded in plastic (JB-4 Embedding Kit, Polysciences), and1–2-mm thick sections (“thin sections”) cut from its proximal, middle,and distal thirds (these segments were arbitrarily designated duode-num, jejunum, and ileum, respectively). Alternatively, after washing in70% ethanol, the intestine was cut open along its duodenal-ileal axis,rolled into a circle, and held in this circular configuration by mountingagar (2% agar (Sigma) in 5% buffered formalin). Each of the resulting“Swiss rolls” was then placed in a tissue cassette, embedded in paraffin,and 5 mm-thick serial sections were prepared. Plastic- or paraffin-embedded sections were stained with hematoxylin and eosin, phloxine/tartrazine, or with Alcian blue and periodic acid Schiff (PAS) usingstandard protocols (37).

Goblet cells were quantitated by counting Alcian blue/PAS-positivecells in all well-oriented jejunal crypt-villus units present in at least twonon-adjacent sections cut from Swiss rolls (sections were prepared fromthree P28 transgenic animals and three normal littermates per pedi-gree). Paneth cells were likewise quantitated by counting phloxine/tartrazine-positive cells in jejunal crypts.

To analyze the distribution of components of the microflora along thecrypt-villus units of specific pathogen-free transgenic animals and theirnormal littermates, mice from the various pedigrees were sacrificed atP28, P42, and P120–P180. Their small intestines were fixed 4–6 h in10% buffered formalin without prior flushing and then cut into 1–2-cmsegments. Each segment was embedded in paraffin, 4–6-mm thicksections were cut, and the sections treated with Warthin-Starry orGram stains (37).

Single and Multilabel Immunohistochemical Analyses—Transgenicmice and their normal littermates were sacrificed at P28, P42, andP120–180 (n 5 3–5/group/pedigree/time point). Some animals receivedan intraperitoneal injection of an aqueous solution of 59-bromo-29-de-oxyuridine (120 mg/kg, BrdU) and 59-fluoro-29-deoxyuridine (12 mg/kg)1.5 to 72 h before sacrifice. The small intestine was then removed fromeach animal, flushed with cold PBS, fixed in Bouin’s solution for 8 h atroom temperature, treated with 70% ethanol, and 4–6-mm thick sec-tions cut from paraffin-embedded Swiss rolls. Sections were then depar-affinized, rehydrated, and placed in PBS-blocking buffer (1% bovineserum albumin, 0.3% Triton X-100 in PBS) for 20 min at room temper-ature. Slides were incubated overnight at 4 °C with the following anti-bodies: (i) rabbit antiserum raised against residues 4–35 of cryptdin-1(the antisera reacts with purified cryptdins 1, 2, 3, and 6 (32, 38), wassupplied by Michael Selsted, University of California, Irvine, and wasdiluted 1:500 in PBS-blocking buffer); (ii) rabbit antiserum to the se-creted phospholipase A2 encoded by Pla2g2a (also known as enhancingfactor; Refs. 20 and 21) was obtained from Rita Mulherkar, CancerResearch Institute, Tata Memorial Center, Bombay, India; dilution 51:40,000); (iii) rabbit anti-human lysozyme (Dako, Santa Barbara, CA;specificity in the FVB/N intestine described in Ref. 32; dilution 51:500); (iv) rabbit anti-serotonin (Incstar, Stillwater, MN; 39; 1:8000);(v) rabbit anti-chromogranin A (Incstar; 1:10,000); (vi) rabbit anti-hGH

1 The abbreviations used are: kb, kilobase pairs; SV40 TAg, simianvirus 40 large T antigen; CR2, nucleotides 26500 to 134 of the mousecryptdin-2 gene; DT-A, diphtheria toxin A fragment; tox176, an atten-uated DT-A containing a Gly128 3 Asp substitution; hGH, humangrowth hormone; P, postnatal day; BrdU, 5-bromo-29-deoxyuridine;PAS, periodic acid-Schiff stain; EPX, endogenous peroxidases; HRP,horseradish peroxidase; FITC, fluorescein isothiocyanate; Cy3, indocar-bocyanine; Cy5, indodicarbocyanine; SFB, segmented filamentous bac-terium; GALT, gut-associated lymphoid system; TCR, T-cell receptor;PBS, phosphate-buffered saline; DBA, Dolichos biflorus agglutinin;UEA-1, Ulex europaeus agglutinin 1.

Effects of Paneth Cell Ablation23730

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

(Dako; 39; 1:2000); (vii) rabbit anti-SV40 TAg (a generous gift of DougHanahan, University of California, San Francisco; 40; 1:2000); and (viii)goat anti-BrdU (Ref. 41; 1:1000). Antigen-antibody complexes weredetected with indocarbocyanine (Cy3)- or indodicarbocyanine (Cy5)-conjugated donkey anti-rabbit or anti-goat immunoglobulins (Ig;Jackson Immunoresearch; 1:500).

Sections were also incubated with a series of fluorescein isothiocya-nate (FITC)-conjugated lectins (all obtained from Sigma, all used at afinal concentration of 5 mg/ml PBS blocking buffer): (i) Ulex europaeusagglutinin 1 (UEA-1; carbohydrate specificity 5 Fuca1,2Gal epitopes;lineage specificity in P28-P180 FVB/N small intestine 5 Paneth, goblet,and enteroendocrine cells; Ref. 42); (ii) peanut (Arachis hypogaea) ag-glutinin (PNA, Galb3GalNAc; all four epithelial lineages; Ref. 42); and(iii) Dolichos biflorus agglutinin (DBA; GalNAca3GalNAc andGalNAca3Gal epitopes; Paneth and goblet cells plus enterocytes; Ref. 42).

The spatial distribution of components of the diffuse gut-associatedlymphoid system (GALT) was examined in P42 CR2-tox176 mice andtheir normal littermates (n 5 3 animals/group/pedigree) using thefollowing panel of monoclonal antibodies from PharMingen (each di-luted 1:1000 in PBS-blocking buffer): (i) rat anti-mouse CD4 (cloneH129.19); (ii) rat anti-mouse a chain of CD8 (clone 53–6.7); (iii) hamsteranti-mouse b-subunit of the ab T-cell receptor (TCR; clone H57–597);(iv) hamster anti-mouse gd TCR (clone GL3); and (v) rat anti-mouseCD45R/B220 (a B-cell marker; clone RA3–6B2). Mice were sacrificed,and the middle third of the small bowel was flushed with PBS and thenfrozen in OCT (Miles). Serial sections were cut, fixed in methanol(220 °C for 15 min), washed 3 times (3 min/cycle) in PBS, and treatedwith PBS-blocking buffer (15 min at room temperature). A variety ofmethods that are traditionally used for eliminating endogenous perox-idase (EPX) activities from cryostat sections of the intestine eitherfailed to adequately remove EPX or to preserve the antigens that wewere studying (cf. Ref. 43). However, we found that endogenous biotinlevels were below the limits of detection with tyramide signal amplifi-cation protocols that employed horseradish peroxidase (HRP)-conju-gated streptavidin. Therefore, cells with EPX activity were labeled byincubating frozen sections of intestine for 8–10 min at room tempera-ture with FITC-conjugated tyramide (obtained from NEN Life ScienceProducts and diluted 1:100 in 1 3 amplification diluent from the samemanufacturer). Following 3 washes in PBS (5 min each), the sectionswere incubated overnight at 4 °C with one of the primary antibodiesand then washed in TNT buffer (0.1 M Tris, pH 7.5, 0.15 M NaCl, 0.05%Tween 20; 3 cycles with 5 min/wash). Two secondary antibodies wereused to visualize antigen-antibody complexes. (i) If HRP-conjugatedgoat anti-rat Ig (Kirkegaard and Perry Labs) was used, it was firstdiluted 1:100 in TNB buffer (TNB 5 0.1 M Tris, pH 7.5, 0.15 M NaCl,0.5% blocking reagent from NEN Life Science Products) and thenplaced on the section for 30 min (this and all subsequent steps wereperformed at room temperature). After three washes with TNT buffer,biotinyl-tyramide was added (diluted 1:100 in 1 3 amplification dilu-ent). Following a 8–10-min incubation, the sections were washed sev-eral times in TNT buffer and overlaid with Cy3-conjugated streptavidin(Jackson Immunoresearch; diluted 1:500 in TNB) for 30 min. (ii) If abiotinylated anti-hamster Ig (diluted 1:100 in TNB) was used as thesecondary antibody, it was detected with HRP-conjugated streptavidin(NEN Life Science Products; diluted 1:1000 in TNB) followed by ampli-fication with biotinyl-tyramide and application of Cy3-streptavidin, asdescribed above.

Two controls were performed for each experiment employing a givenprimary antiserum: (i) direct amplification of EPX alone (see above) and(ii) omission of primary antibodies. The latter control involved directamplification of EPX followed by application of an HRP-conjugatedsecondary antibody and subsequent indirect tyramide signal amplifica-tion with biotinyl tyramide and Cy3-streptavidin. Alternatively, whenbiotinylated secondary antibodies were employed, there would be acontrol to establish whether there was any labeling of endogenousbiotin. This control consisted of direct tyramide amplification of EPXwith FITC-tyramide followed by HRP-streptavidin but without additionof the biotinylated secondary antibodies or the primary antibodies.(Note that adding Cy3-streptavidin alone to jejunal sections did notproduce any cellular staining.)

Light Microscopy—A Molecular Dynamics Multiprobe 2001 invertedconfocal microscope system was used to scan sections subjected to singleand/or multi-label immunohistochemistry. Sections were also viewedand photographed using a Zeiss Axioscope.

Identification of Apoptotic Cells—Apoptotic cells were scored in ad-jacent sections of Swiss rolls, prepared from normal, CR2-tox176, andCR2-TAg mice, using the terminal deoxynucleotidyltransferase-medi-ated, dUTP nick end-labeling assay, and by their morphologic appear-

ance after staining with hematoxylin and eosin (6, 7, 44). Incorporationof digoxigenin-labeled dUTP was detected using peroxidase-conjugatedsheep anti-digoxigenin Fab fragments (Boehringer Mannheim, diluted1:500 in PBS-blocking buffer) and the Vector VIP kit (Vector Labora-tories). Sections were counterstained with methyl green (Zymed).

EM Morphologic Analysis—Three-mm2 fragments were obtainedfrom the distal jejunum of CR2-tox176 and CR2-TAg transgenic miceplus their age-matched littermates (n 5 two P28 animals/pedigree).(Note that “distal jejunum” was defined as two-thirds of the distancefrom the gastro-duodenal junction to the ileal-cecal junction.) The frag-ments were then fixed for 6 h at 4 °C in 2% paraformaldehyde, 2%glutaraldehyde (prepared in PBS), washed in PBS, post-fixed for 1 h in2% osmium tetroxide, and stained with a solution containing aqueousuranyl acetate and lead. Samples were dehydrated in graded alcoholsand embedded in Poly/Bed 812 (Electron Microscopy Sciences). Onehundred nanometer-thick sections were prepared and viewed with aJOEL model 100C electron microscope.

EM Immunohistochemical Analysis—Fragments from the distal je-junum were fixed as above, washed with PBS, dehydrated with gradedethanols, and embedded in Lowicryl (Polysciences). Fifty to seventynanometer-thick sections were cut, placed on 100-mesh Formvar-coatedgrids (Electron Microscopy Sciences), and floated for 30 min at roomtemperature on a solution of Tris-buffered saline-blocking buffer (20mM Tris, 150 mM NaCl, pH 7.4, 10% normal mouse serum, 0.3% Tween20). Grids were then incubated for 2 h at room temperature with rabbitanti-mouse cryptdin (see above; diluted 1:50 with Tris-buffered saline,5% normal mouse serum, 0.3% Tween 20), rabbit anti-mouse Pla2g2a(1:4000), or rabbit anti-hGH (Dako, 1:100; 45). Following washes withTris-buffered saline, 0.3% Tween 20, antigen-antibody complexes weredetected with 18-nm diameter colloidal gold-conjugated goat anti-rabbitIgG (Jackson Immunoresearch, diluted 1:15). Grids were counter-stained with aqueous uranyl acetate and lead.

RESULTS

General Comments About Paneth, Goblet, and IntermediateCells—Paneth cells are distributed along the length of theduodenal-ileal axis in adult (postnatal day 28 to 180) FVB/Nmice (32). They can be recognized based on staining of theircharacteristic apical secretory granules with tartrazine, bytheir reaction with antibodies directed against lysozyme, thesecreted phospholipase encoded by Pla2g2a, and cryptdins, aswell as by their production of fucosylated glycoconjugates de-tected by the lectins Urex europaeus agglutinin type 1 (UEA1),peanut (Arachis hypogaea) agglutinin (PNA), and Dolichosbiflorus agglutinin (DBA) (32, 42).

Approximately 10% of goblet cells in the normal adult mouseintestine contain very small electron dense cores within theirmucin granules. As these “granule goblet cells” migrate up villiand differentiate, they secrete their dense core mucin granuleswhich are then replaced by “common” mucin granules that lackelectron-dense cores (8). A rare cell type has been observed innormal small intestinal crypts. Its granules contain electron-dense cores that are intermediate in size between those ingranule goblet cells and those in the apical granules of youngPaneth cells (Fig. 1, A–C). The granules of these rare cells alsocontain small amounts of mucin. Because of their morphologicfeatures, they have been termed “intermediate” (13), “granulo-mucous” (46), or “transitional” (47, 48) cells. These cells havebeen proposed to be Paneth cells undergoing transformation togoblet cells, goblet cells in the process of being converted toPaneth cells, or a precursor of both lineages.

Nucleotides 26500 to 134 of the Mouse Cryptdin-2 Gene AreActive in the Paneth, Granule Goblet, and Intermediate Cells ofAdult Transgenic Mice—Light microscopic surveys of adultFVB/N small intestine disclosed that ;95% of all crypts pres-ent in a cross-section contain cryptdin-positive cells (average 53 cells/duodenal crypt section; 5 cells/ileal crypt section). EMimmunohistochemical analysis using polyclonal antibodiesthat recognize several members of the cryptdin family revealedthat cryptdins are present in the dense core granules of granulegoblet cells, intermediate cells, and Paneth cells (Fig. 1, E and

Effects of Paneth Cell Ablation 23731

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

F). The EM study also indicated that these cryptdins are notexpressed in any other intestinal epithelial cell type, includingmature goblet cells (data not shown).

We subsequently used light and EM immunohistochemistryto define the small intestinal patterns of expression of a humangrowth hormone reporter in several pedigrees of P28 to P180transgenic mice containing a cryptdin-226500 to 134/humangrowth hormone fusion gene (CR2-hGH). The results estab-lished that nucleotides 26500 to 134 of the mouse cryptdin-2gene are active in Paneth, granule goblet, and intermediatecells (e.g. Fig. 1D) and silent in all other epithelial cell typespresent in crypt-villus units.

Cryptdin-226500 to 134-directed Expression of an AttenuatedDiphtheria Toxin A Fragment Results in Paneth Cell Abla-tion—Fragment A of diphtheria toxin (DT-A) ADP-ribosylatesand inactivates elongation factor 2, causing inhibition of pro-tein synthesis and cell death. tox176 is an attenuated DT-Aallele with a Gly1283 Asp substitution that reduces its potency15–30-fold (35). The attenuated toxin was chosen for Panethcell ablation in transgenic mice because it is less likely than

wild type DT-A to cause death if expressed at low basal levelsin nontarget cell populations (49).

Two pedigrees of FVB/N cryptdin-226500 to 134/tox-176 (CR2-tox176) mice were produced. There were no statistically signif-icant differences between the growth rates and adult bodyweights of CR2-tox176 mice and their normal littermates. Com-parably aged members of each pedigree had identical pheno-types (see below).

FIG. 2. Phloxine/tartrazine histochemical stains of FVB/N nor-mal, CR2-tox176, and CR2-TAg transgenic small intestine. 1-mmthick sections were prepared from plastic-embedded sections of thedistal jejunum of P28 normal or transgenic mice and stained withphloxine and tartrazine (P/T). A, crypt-villus units from a nontrans-genic littermate. Note that Paneth cells contain distinctive apical tar-trazine-positive granules (red). These cells are confined to the base ofcrypts. B, high power view of a crypt from the animal in A. The solidarrow points to the tartrazine-positive apical secretory granules of aPaneth cell. C, high power view of several crypts from a P28 CR2-tox176mouse. Note the absence of Paneth cells detectable by this histochem-ical stain. The solid arrows point to several apoptotic cells that can bevisualized with tartrazine or by dUTP nick end-labeling assay of anadjacent section. As expected from the known mechanism of action ofDT-A, there was increased cell death in the crypts of transgenic micecompared with their normal littermates. Comparison of A and B with Cemphasizes the extent of the Paneth cell ablation produced by theattenuated DT-A. D, high power view showing several crypts from aCR2-TAg mouse. Ablation of Paneth cells is evident as are severalapoptotic cells (arrows). Bars 5 25 mm.

FIG. 1. EM immunohistochemical analysis of the cellular pat-tern of expression of CR2-hGH in adult FVB/N transgenic mice.A–C, transmission EM of crypt epithelial cells from the distal jejunumof a normal P28 FVB/N mouse. The distal jejunum is defined as thejunction between the middle and distal thirds of the small intestine. A,view of the lower portion of the crypt containing Paneth cells (e.g.arrow) with their electron-dense apical secretory granules. Bar 5 10mm. B, transmission EM of the supranuclear region of an intermediatecell from the same mouse. Characteristic mucin-containing secretorygranules with electron dense cores are found in this region of the cell.Bar 5 3 mm. C, supranuclear region of a Paneth cell showing details ofsecretory granule morphology. Bar 5 3 mm. D, immunohistochemicalevidence of hGH expression in the mature Paneth cells of a P28 CR2-hGH transgenic mouse. The section was incubated with rabbit anti-hGH and gold-conjugated goat anti-rabbit Ig. The 18-nm diameter goldparticles are evident in the electron dense apical secretory granules.Bar 5 1 mm. E and F, cryptdins are present in the dense core granulesof intermediate cells (E) and granule goblet (F) cells in a normalFVB/N P28 mouse. The section was incubated with rabbit polyclonalantibodies that react with several members of the cryptdin family.Antigen-antibody complexes were visualized with gold-labeled second-ary antibodies as in D. Bar 5 1 mm.

Effects of Paneth Cell Ablation23732

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

A 95% reduction in the number of Paneth cells was evidentthroughout the length of the small intestines of transgenicanimals by P28, whether defined by a loss of staining of serialsections with tartrazine (Fig. 2, A–C), a loss of cellular reactiv-ity with the lectins UEA-1, PNA, and DBA, or the failure ofantibodies to detect cryptdins, lysozyme, and Pla2g2a (phos-pholipase A2) in crypt-villus units (e.g. Fig. 3, A and B). Theablation of Paneth cells was verified using transmission EM(Fig. 4A).

The reduction in Paneth cell number within the small intes-tines of CR2-tox176 mice persists from P28 through at leastP180, although the magnitude of the reduction is less at latertime points (e.g. 82% at P42). Granule goblet and intermediatecells can only be defined using EM methods that are not usefulfor broad surveys of rare cell populations. Nonetheless, EMstudies indicated that these cells were reduced in number, butnot as markedly as Paneth cells.

The space normally occupied at the crypt base by Panethcells was occupied in CR2-tox176 mice by “crypt base columnarcells” (Fig. 4, A–C). Crypt base columnar cells are normallyinterspersed among Paneth cells and constitute 60–70% of thecells that populate the bottom three cell layers of duodenal,jejunal, and ileal crypts (9). Previous [3H]thymidine label-ing/EM radioautography studies indicated that: (i) their resi-dence time at the crypt base after entering S-phase is just a fewhours; (ii) they migrate up and out of duodenal, jejunal, andileal crypts within 3–4 days; and (iii) they differentiate intoenterocytes (9). The morphologic features of crypt base colum-nar cells present in CR2-tox176 transgenic mice and in theirnormal littermates were indistinguishable. In each case, theyresembled the undifferentiated, proliferating transit cell popu-lation located in the mid-crypt (see Fig. 4C and legend).

To define the effects of Paneth cell ablation on epithelial cellproliferation, P28, P42, and P120–180 CR2-tox176 mice andtheir age-matched normal littermates were pulse-labeled withBrdU 1.5 h prior to sacrifice. The number of S-phase cells wascounted in sections of distal jejunal crypts (n 5 3 mice/pedigree/time point). There were no statistically significant differencesbetween the total number of S-phase cells in the middle andupper thirds of normal and CR2-tox176 transgenic crypts.Since the fractional representation of crypt base columnar cells

FIG. 3. Distal jejunal crypt-villus units from age-matched nor-mal, CR2-tox176, and CR2-TAg transgenic FVB/N mice, showingthe cellular patterns of expression of cryptdins andFuca1,2Galb-containing glyconjugates. A, confocal micrograph ofcrypt-villus units from a normal P28 FVB/N mouse that had beentreated with BrdU 1.5 h prior to sacrifice. The section was incubatedwith goat anti-BrdU and rabbit anti-cryptdins (detected with Cy3-donkey anti-goat Ig and Cy5-donkey anti-rabbit Igs, respectively), plusFITC-conjugated UEA-1. The solid arrow points to a UEA-1-positivevillus goblet cell (green). Open arrows point to UEA-1- and cryptdin-positive Paneth cells (blue/green) at the base of the crypts. Epithelialcells with BrdU-positive nuclei (red) are limited to the crypt. Crypt-villus junctions are denoted by closed arrowheads in this and subse-quent panels. B, confocal micrograph of crypt-villus units from a P28CR2-tox176 littermate of A that had also been pulse-labeled with BrdU1.5 h prior to sacrifice. The section was stained as in A. Paneth cells are

absent as judged by the lack of UEA-1- or cryptdin-positive cells at thecrypt base. BrdU-positive crypt base columnar cells (e.g. open arrow)occupy the space where Paneth cells normally reside. C and D, standardlight micrographs of crypt-villus units from a P28 normal mouse (C) andits CR2-tox176 littermate (D). Mice received BrdU 48 h prior to sacri-fice. The sections were stained with goat anti-BrdU (detected as redwith Cy3-donkey anti-goat Ig) and rabbit anti-cryptdin (visualized asdark blue with AMCA-conjugated donkey anti-rabbit Ig). The leadingand trailing edges of the columns of BrdU-positive cells are comparablypositioned in normal and transgenic mice, indicating that loss of Panethcells does not affect cell movement and that crypt base columnar cellsare able to exit the crypt along with former members of the transit cellpopulation. E, confocal micrograph of crypts from a P28 CR2-TAgmouse. The section was incubated with rabbit anti-SV40 TAg (detectedas orange with Cy3-donkey anti-rabbit Ig) and FITC-UEA1. The openarrow points to a crypt devoid of UEA1-positive Paneth cells. The closedarrow points to a SV40 TAg-negative, UEA-1-positive Paneth cell in theadjacent crypt. Paneth cells were rarely encountered in these mice.When present they were invariably SV40 TAg-negative. F, confocalmicrograph of crypt-villus units from a P28 CR2-TAg mouse that hadbeen labeled 1.5 h before sacrifice with BrdU. The section was incu-bated with antibodies to BrdU (visualized as red with Cy3-donkeyanti-goat Ig), FITC-conjugated UEA1 (green), and SV40 TAg (detectedas blue with Cy5-donkey anti-rabbit Ig). Villus epithelial cells ex-pressing SV40 TAg are UEA-1-positive. Members of this amplifiedgoblet cell lineage contain purple nuclei (e.g. solid arrows) indicatingthat they had entered S-phase within the 1.5-h period before sacrifice.Open arrows point to BrdU-positive, SV40 TAg-negative crypt basecolumnar cells. Bars 5 25 mm.

Effects of Paneth Cell Ablation 23733

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

was markedly increased in transgenic mice, it was not surpris-ing that there was a greater number of BrdU1 cells in the lowerthird of transgenic crypts (Fig. 3B).

Surveys of sections of duodenal, jejunal, and ileal villi pre-pared from normal and CR2-tox176 mice that had receivedBrdU 1.5 h before sacrifice failed to reveal villus epithelial cellsin S-phase (Fig. 3B). Transgenic mice and their normal litter-mates were subsequently sacrificed at later time points afterreceiving BrdU. There were no differences in the rate of clear-ance of BrdU-positive cells from normal or transgenic crypts;by 24 h, BrdU1 cells were no longer present in the lowertwo-thirds of duodenal, jejunal, or ileal crypts; by 36 h, cryptswere free of these labeled cells; and by 48 h and 72 h, thelocation of the trailing edge of BrdU1 cells on duodenal, jejunal,or ileal villi was similar in normal and transgenic animals (e.g.Fig. 3, C and D; (n 5 2–6 animals/time point/pedigree)). Theseresults indicate that the columnar cells that populate the baseof transgenic crypts are not abnormally retained at this loca-tion in the absence of Paneth cells. The data are also consistentwith the notion that the production and subsequent upwardmigration of crypt epithelial cells are not grossly perturbed bythe lineage ablation.

Loss of Paneth cells had no demonstrable qualitative orquantitative effects on the other three small intestinal epithe-lial cell lineages. A comparison of CR2-tox176 mice and theirage-matched normal littermates did not reveal any statisticallysignificant differences in the number of their Alcian blue/PAS-positive goblet cells per duodenal, jejunal, or ileal villus section(e.g. Fig. 5, A–C). The goblet cell lineage normally exhibits

complex variations in its pattern of glycoconjugate productionalong the crypt-villus and duodenal-ileal axes. These variationsare a sensitive marker of the lineage’s differentiation programand are definable with a panel of lectins and in situ histochem-ical assays (42). Histochemical surveys using three of theselectins (UEA1, DBA, and PNA) indicated that terminal differ-entiation of goblet cells is unaffected by loss of Paneth cells(Fig. 3A, B plus data not shown). Based on results obtainedwith antibodies to chromogranin A and serotonin plus the threelectins, we concluded that there were no significant changes inenteroendocrine cell number or differentiation. The enterocyticlineage also appears unaffected, as judged by the number anddistribution of villus epithelial cells that react with antibodiesto intestinal fatty acid binding protein and with lectins thatrecognize fucosylated glyconjugates (data not shown).

Loss of Paneth Cells Does Not Have Any Appreciable Effect onthe Crypt-Villus Distribution of Components of the Microflora—Paneth cells are not present in adult FVB/N colonic crypts.Bacterial density increases along the duodenal-colonic axis ofmice and man (50). The colonic crypts of many mammals,including mice, are colonized by bacteria. Microbial coloniza-tion is rarely seen in the small intestinal crypts of healthyanimals. The secretion of cryptdins and lysozyme from theapical secretory apparatus of Paneth cells may help preventcolonization.

We used a prominent and easily detectable member of thenormal FVB/N intestinal microflora to determine whether lossof Paneth cells affected the distribution of bacteria along thecrypt-villus and duodenal-ileal axes. A segmented filamentousbacterium (SFB), thought to belong to the Gram-positive Clos-tridia (51), colonizes the normal small intestine after weaning(52) and reaches very high densities in the ileum of adultanimals. The SFB cannot be recovered from the intestine andcultured in vitro (53, 54), but can be easily seen in the ileum ofnormal FVB/N mice using the Warthin-Starry silver stain. SFBadheres to ileal enterocytes located in the upper half of thevillus (Fig. 6, A and B). It is not present in crypts (Fig. 6A).Unperfused small intestines from P28 CR2-tox176 transgenicmice and their normal littermates (housed in the same mi-croisolator cages) were subdivided into 1–2-cm segments alongthe length of the duodenal-ileal axis. Warthin-Starry stains ofsections prepared from each segment indicated that loss ofPaneth cells had no effect on the duodenal-ileal or crypt-villusdistribution of SFB (Fig. 6, A and C). Gram stains providedindependent confirmation that duodenal, jejunal, and ilealcrypts of P28-P180 CR2-tox176 mice were free of detectablemicrobes (data not shown).

Ablation of Mature Paneth Cells Is not Associated with aChange in the Crypt-Villus Distribution of Components of theDiffuse Gut-associated Lymphoid Tissue (GALT)—Luminal an-tigens and microbes are delivered by M-cells to submucosallymphoid tissues (e.g. Peyer’s patches). These components ofthe organized GALT can serve as inductive sites for initiationof immune responses (56). Analysis of intestines harvestedfrom specific pathogen-free P28-P180 transgenic mice and theirnormal littermates indicated that loss of Paneth cells is notaccompanied by changes in the size, number, distribution, orhistochemical features of Peyer’s patches (data not shown).

Complex, dynamic, and often subtle interactions occur be-tween the gut epithelium and components of the diffuse GALT.For example, mice homozygous for a null allele of the g subunitgene of the T-cell receptor (TCR) lack gd T-cells and exhibit areduction in crypt cellularity as well as a reduction in epithelialcell migration rates up the villus. Mice homozygous for a nullallele of the b subunit gene of the TCR lack ab T-cells but donot manifest these abnormalities (57).

FIG. 4. Transmission EM of a distal jejunal crypt from a P28CR2-tox176 mouse showing ablation of the Paneth cell lineage.A, section showing the entire crypt. The base of the crypt is occupied bypoorly differentiated columnar epithelial cells that lack secretory gran-ules. Bar 5 4 mm. B, higher power view of three adjacent crypt basecolumnar cells from this mouse. Bar 5 1 mm. C, two crypt base colum-nar cells adjacent to an enteroendocrine cell. The closed arrowheadpoints to a secretory granule in this enteroendocrine cell located on theright-hand portion of the panel. The centrally positioned, cylindricallyshaped crypt base columnar cell contains a basal nucleus, a plasmamembrane that is smooth and does not interdigitate with adjacent cells,numerous supranuclear mitochondria (e.g. closed arrows), and a prom-inent supranuclear Golgi apparatus (e.g. open arrows). Bar 5 1 mm.

Effects of Paneth Cell Ablation23734

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

The normal distribution of components of the diffuse GALTalong the crypt-villus axis has not been extensively character-ized because of the difficulty in identifying cellular markerswith conventional immunohistochemical detection methods.We used a protocol employing tyramide signal amplification toidentify these components in serial sections of jejunum pre-pared from age-matched FVB/N CR2-tox176 mice and theirnormal nontransgenic cagemates.

In normal P42 animals with a conventional microflora (n 53), CD41 T-cells are largely confined to the lamina propria andare present throughout the length of the crypt-villus axis (Fig.7B). CD81 intraepithelial T-cells have been postulated to servecytotoxic as well as immunosuppressive functions (58). In nor-mal P42 animals, CD81 T-cells, unlike CD41 T-cells, are pre-dominantly intraepithelial and restricted to the villus (Fig.7D). ab T-cells are distributed along the length of the crypt-villus axis and populate the intraepithelial and lamina propriacompartments (Fig. 7F). In contrast, gd T-cells are limited tothe villus where they are predominantly located within theepithelium (Fig. 7H). B-cells, defined using CD45R/B220 as a

marker, are confined to the lamina propria and are distributedfrom the base of the crypt to the villus tip (data not shown).These results are summarized in Fig. 7A.

Comparable immunohistochemical surveys of P42 CR2-tox176 mice (n 5 3) indicated that ablation of Paneth cells wasnot associated with any detectable alteration in the distribu-tion of these components of the diffuse GALT (Fig. 7, C, E, G,and H). Moreover, hematoxylin and eosin stains of sectionsprepared along the length of P28-P180 CR2-tox176 small in-testines failed to disclose any evidence of acute or chronicinflammatory changes (n 5 40 animals). Together, these re-sults suggest that Paneth cells do not have a direct or indirectorganizing function for the diffuse GALT. Furthermore, theyare consistent with the notion that loss of Paneth cells does notproduce marked perturbations in host-microbial interactions inpathogen-free mice.

Cryptdin26500 to 134-directed Expression of SV40 TAg BlocksPaneth Cell Differentiation but Amplifies Intermediate andGranule Goblet Cells—Promoter-targeted expression of simianvirus T antigen (SV40 TAg) in the progenitor cells of specific

FIG. 5. Alcian Blue/PAS stain of goblet cells in distal jejunal crypt-villus units from P28 normal and transgenic mice. A, section froma normal FVB/N mouse. Goblet cells with their characteristic mucin globules are scattered along the villus (e.g. closed arrow). B, higher power viewof one of the villi shown in A. C, crypt-villus units from a CR2-tox176 littermate. Goblet cell number is similar to that observed in its normallittermate (compare with A). D, crypt-villus units from a FVB/N P28 CR2-TAg mouse. The number of goblet cells is markedly increased relativeto normal FVB/N or CR2-tox176 animals. E, higher power view of one of the villi shown in D. Bars 5 25 mm.

FIG. 6. Distribution of segmented filamentous bacteria along the crypt-villus axis of normal and transgenic mice. A, Warthin-Starrysilver stain of a section containing proximal ileal crypt-villus units from a normal P28 FVB/N mouse (ileum is defined as the distal third of the smallintestine). Arrows point to SFB adhering to the tips of villi. Crypts are not colonized with this organism or other components of the microfloradetectable with the histochemical stain. B, transmission EM showing three SFB interacting with a normal FVB/N ileal villus enterocyte. Theenterocyte’s apical microvilli are intact. The enterocyte develops an actin-containing, electron-dense area (arrow) around the intruding head piece(see Ref. 55). Bar 5 1 mm. C, ileal crypt-villus units from a P28 CR2-tox176 transgenic mouse. Loss of Paneth cells does not result in colonizationof crypts. Bars in A and C 5 25 mm and in B 5 1 mm.

Effects of Paneth Cell Ablation 23735

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

lineages has been exploited to generate transgenic mouse mod-els of specific physiologic deficiency syndromes. Physiologicdeficiencies result because differentiation of these progenitorsis blocked; the “entrapped” progenitors do not have the func-tional capacities of their terminally differentiated descendantsand therefore cannot compensate for their loss (59). SV40 TAg-stimulated amplification of normally rare progenitors alsoprovides an opportunity to study their intrinsic propertiesand/or the consequences of their increased representation(e.g. Refs. 59 and 60).

With these thoughts in mind, nucleotides 26500 to 134 wereused to direct expression of SV40 TAg in FVB/N transgenicanimals. Three lines of CR2-TAg mice were analyzed from P28to P180. All pedigrees had identical intestinal phenotypes. Thegrowth rates and adult body weights of transgenic mice werenot significantly different from those of their normallittermates.

Staining with tartrazine, UEA1, and antibodies to cryptdins,lysozyme, and enhancing factor revealed a decrease in thenumber of mature Paneth cells in CR2-TAg mice comparable tothe decrease observed in similarly aged CR2-tox176 animals(e.g. 90–95% at P28) (Figs. 2D and 3F). EM confirmed thePaneth cell ablation (Fig. 8A). The few mature Paneth cellsobserved in scattered duodenal, jejunal, and ileal crypts did notcontain detectable levels of SV40 TAg (Fig. 3E). The mousecryptdin gene family includes at least 17 members whose ex-

pression varies both as a function of developmental stage andcellular position along the crypt-villus and duodenal-ileal axes(30). Although we do not have antibodies specific for cryptdin-2,it seems likely that this small population of residual matureSV40 TAg-negative Paneth cells was able to complete its ter-minal differentiation because its members do not support ex-pression of the endogenous cryptdin-2 gene or transgenes un-der the control of cryptdin-226500 to 134.

SV40 TAg-positive epithelial cells were distributed along thelength of the crypt-villus axis. These cells were most abundantin the crypts and lower half of the villus. SV40 TAg levelsdecreased as cells moved to the upper half of the villus(Fig. 3F). SV40 TAg-positive villus epithelial cells were alsoUEA-1-positive (Fig. 3F). The UEA-1/SV40 TAg-positive cellswere members of the goblet cell lineage. Unlike CR2-tox176mice, CR2-TAg animals exhibit a statistically significant 2–3-foldincrease in the number of Alcian blue/PAS-positive goblet cellsper duodenal, jejunal, or ileal villus section (p , 0.05; referencecontrol 5 age-matched normal littermates) (Fig. 5, D and E).Pulse labeling with BrdU 1.5 h prior to sacrifice revealed thatproduction of SV40 TAg is associated with re-entry of thesevillus goblet cells into S-phase (Fig. 3F).

EM immunohistochemical studies provided further insightsabout the origins of this amplified goblet cell population. Anal-yses of distal jejunal crypts from CR2-TAg mice and theirnormal littermates disclosed a marked amplification of crypt-

FIG. 7. Ablation of the Paneth celllineage in CR2-tox176 is not associ-ated with changes in the crypt-villusdistribution of components of the dif-fuse GALT. A, schematic representationof results obtained from immunohisto-chemical surveys of jejunal sections pre-pared from P42 FVB/N normal mice andtheir CR2-tox176 (tox) transgenic litter-mates. B–H, frozen sections were pro-cessed as described under “ExperimentalProcedures.” Note that in each panel ofthe figure, antigen-antibody complexeshave been detected using HRP-conju-gated secondary antibodies, biotinyl tyra-mide amplification, and Cy3-streptavidin.Cells that react with the primary antibod-ies and have no endogenous peroxidaseactivity appear red-orange. Cells that con-tain endogenous peroxidase activity areco-labeled with FITC-tyramide and Cy3-streptavidin and therefore appear yellow-green. B and C, CD41 T-cells (red-orange)are largely confined to the lamina propriaand are present throughout the length ofthe crypt-villus axis in normal (B) andtransgenic (C) animals. Solid arrows in-dicate the location of crypt-villus junc-tions, and open arrows point to the base ofcrypts. D and E, CD81 T-cells populatethe lamina propria and intraepithelialcompartments of the villus but are absentfrom the crypt in normal (D) and trans-genic (E) mice. F and G, distribution ofT-cells expressing the ab T-cell receptor(TCR) in normal (F) and transgenic (G)littermates. H, gd TCR cells populate theintraepithelial compartment of the villusand are absent from the crypt of thistransgenic mouse. An identical distribu-tion was observed in normal littermates(data not shown). Bars 5 25 mm.

Effects of Paneth Cell Ablation23736

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

din- and phospholipase A2 (Pla2g2a)-positive intermediate andgranule goblet cells in transgenic crypts (Fig. 8, A–G). Inter-mediate cells were found in the lower two-thirds of thesecrypts. The diameter of the granule’s electron-dense core di-minishes, and the relative area occupied by its mucin increasesas cryptdin- and phospholipase A2-positive cells occupy theupper regions of transgenic crypts, resulting in an amplifiedpopulation of granule goblet cells positioned in the upper crypt/lower villus (Fig. 8, A–G). SV40 TAg- and UEA1-positive gobletcells in the upper half of the villus lack dense core granules, donot contain the secreted phospholipase A or cryptdin, and havethe morphologic, histochemical, and immunohistochemical fea-tures of normal mature common goblet cells (data not shown).These results suggest a sequence of “differentiation” involvingtransformation from intermediate to granule goblet to “ma-ture” common goblet cell.

Even though the ablation of mature Paneth cells by CR2-TAgwas accompanied by an amplification of intermediate and gran-ule goblet cells, the crypt base in these mice resembled thecrypt base in CR2-tox176 animals, i.e. the position normallyoccupied by mature Paneth cells was partially filled by cryptbase columnar cells (Fig. 8A). These crypt base columnar cellslack SV40 TAg but incorporate BrdU (Fig. 3F).

Other Phenotypic Considerations—Macro- and microscopicsurveys of the intestines of P28–P180 mice from the three

CR2-TAg pedigrees indicated that expression of this viral on-coprotein did not lead to the development of intestinal neo-plasms (n 5 40 animals). The increased levels of apoptosisnoted in the crypts of CR2-TAg mice (Figs. 3D and 8A) suggestthat some differentiating Paneth cells could be cleared by thismechanism, induced as a consequence of their SV40 TAg-induced proliferation. There is precedent for this elsewhere inthe crypt-villus axis; forced expression of SV40 TAg in post-mitotic FVB/N villus enterocytes using a different promoterresults in their pRB-dependent re-entry into the cell cycleand the induction of a p53-independent apoptosis (61). Aug-mented apoptosis of SV40 TAg1 Paneth cells may explain, inpart, why CR2-TAg mice do not develop intestinal neoplasms(see “Discussion”).

As with CR2-tox176 animals, there were no apparent per-turbations in host-microbial interactions. Gram and Warthin-Starry stains revealed no detectable crypt colonization and nochange in the spatial distribution of segmented filamentousbacteria compared with their normal littermates or to CR2-tox176 animals (data not shown). CR2-TAg animals do notdevelop evidence of gut mucosal inflammation (n 5 40 mice,28–180 days old).

DISCUSSION

Paneth Cells Do Not Appear to Be Necessary to Establish andMaintain a Functional Stem Cell Niche—The precise locationof the multipotent stem cell in the adult mouse small intestinalcrypt has not been established. However, tritiated thymidinelabeling/radioautographic analyses of cell proliferation, move-ment, and differentiation programs have led to speculationthat stem cells are positioned in the fifth cell stratum from thecrypt base (1–3, 5).

Immature Paneth cells are located just above and below thepresumptive stem cell niche. They differentiate during a down-ward migration to the crypt base where they comprise ;50% ofthe cell population in the first through fourth cell layers (9).The location and direction of Paneth cell migration, the con-comitant acquisition of the ability to produce growth factors,their potential for providing instructions to neighboring cellsduring their downward descent, and their high density and thelong residency time at the crypt base raise the possibility thatPaneth cells may influence the structure and/or function of thestem cell niche. Paneth cell ablation by an attenuated diphthe-ria toxin A fragment represents the first reported experimentalmanipulation of the cellular microenvironment that purport-edly contains the stem cell and its immediate descendants.

The gut provides a unique system in which to perform thesetypes of experimental manipulations because the stem cell iscontained in a readily detectable anatomic unit, i.e. the crypt.Furthermore, each adult mouse crypt contains a monoclonalpopulation of cells (62–64). The stem cell’s descendants un-dergo 4–6 rounds of rapid cell division, generating a steadystate population of ;250 crypt epithelial cells of which ;150are cycling at any given moment (5). Thus, perturbations thatchange the biological properties of the stem cell and its imme-diate descendants may be inferred by noting changes in thebehavior and/or composition of their amplified progeny.

The results of tox176-mediated ablation of Paneth cells sug-gest that this lineage is not essential for establishment of afunctional stem cell niche, at least based on the observationsthat (i) proliferative activity is maintained in crypts over thefirst 6 months of life and (ii) the composition of terminallydifferentiated members of the intestine’s other self-renewingepithelial lineages is not perturbed. Further analysis of thelonger term effects of Paneth cell loss in this model is limited bythe gradual re-appearance of members of this lineage. Thiscould reflect time-dependent changes in CR2-mediated expres-

FIG. 8. CR2-TAg mice contain an amplified population ofcryptdin- and phospholipase A2-producing intermediate andgranule goblet cells. A, transmission EM of a distal jejunal cryptshowing ablation of mature Paneth cells. Intermediate cells are ampli-fied (e.g. solid arrowheads) as are granule goblet cells (e.g. open arrow-head). The open arrow points to an apoptotic cell. Bar 5 4 mm. B–D, EMimmunohistochemical demonstration of Pla2 in the dense core granulesof intermediate cells located in the lower and middle thirds of the crypt(D and C, respectively) and in a granule goblet cell from the upper thirdof the crypt (B). The sections were incubated with rabbit anti-phospho-lipase A2 (Pla2g2a) and gold-labeled goat anti-rabbit Ig. Bar 5 1 mm.E–G, EM immunohistochemical demonstration of cryptdin accumula-tion in the secretory granules of intermediate cells located in the lowerand middle thirds of the crypt (G and F, respectively), and in a granulegoblet cell positioned in the upper crypt (E). Sections were treated withrabbit anti-cryptdin and gold-labeled goat anti-rabbit Ig. Note how thediameter of the electron dense cores diminishes as cells move up thecrypt-villus unit. As long as these cores are present, cryptdin andenhancing factor are detectable. Control experiments using non-im-mune serum gave no signal (data not shown). Villus goblet cells withcommon mucin globules that lack electron dense cores do not containdetectable levels of the phospholipase A2 or cryptdin (data not shown).Bars 5 1 mm.

Effects of Paneth Cell Ablation 23737

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

sion of tox176. If the level rather than the generality of CR2-tox176 expression in Paneth cells is the cause of this phenom-enon, then using wild type DT-A may produce a longer lastingablation.

Changes at the crypt base in CR2-tox176 animals includeloss of mature Paneth cells and an increase in the proportion ofcrypt base columnar cells. Therefore, we can also conclude thatthe augmented representation of crypt base columnar cellsdoes not have a demonstrable effect on the functional proper-ties of the gut stem cell.

We do not know whether ablation of Paneth cells is associ-ated with a change in the physical location of stem cells in thecrypt or whether that location is fixed even in normal animals.Although the properties of “stemness” in the crypt may not bedefined by instructive interactions involving neighboring Pan-eth cells, signals derived from other neighboring epithelial cellpopulations and/or components of the underlying extracellularmatrix/mesenchyme may play critical roles.

Directional Migration and the Differentiation of Paneth andGoblet Cell Lineages—The first through fourth cell strata innormal adult mouse crypts also contain a small percentage(1–2%) of oligomucous and intermediate cells (9). A relation-ship between Paneth and goblet cell differentiation has beensuggested but never proven. The morphologic features of inter-mediate cells had suggested to others that they may be veryyoung granule goblet cells or a precursor of differentiated Pan-eth and granule goblet cells (13). To explore this possible pre-cursor-product relationship, various groups have attempted todetect lysozyme in intermediate cell granules by light micro-scopic immunohistochemistry. Some workers have reportedsuccess (65), others failure (66, 67). Because of the high back-ground of nonspecific cellular staining observed when commer-cially available antibodies to lysozyme were used for EM im-munohistochemistry, we have also been unable to determinewhether intermediate cells contain lysozyme.

Although lysozyme has long been considered part of theimmunohistochemical definition of a Paneth cell, cryptdins andthe secreted phospholipase A2 encoded by Pla2g2a have beenrecently identified as markers of this lineage. We have nowdemonstrated that cryptdins are produced in the intermediateand granule goblet cells of normal adult FVB/N small intestinalcrypts. In addition, cells with morphologic and immunohisto-chemical features of the ordinarily rare crypt intermediate cellsabound in CR2-TAg crypts. These intermediate cells produceSV40 TAg, proliferate, and support expression of cryptdins aswell as the phospholipase A2. As these SV40 TAg-positive cellsemerge from the crypt and move up the villus, they undergo asequence of cellular alterations that includes a decrease in thesize and number of their electron dense granules and an in-crease in their mucin content. Immunoreactive cryptdins andthe phospholipase A2 are retained until these granules are lostand cellular SV40 TAg expression is suppressed in mature orcommon goblet cells.

These observations suggest that SV40 TAg is expressed in anintermediate cell precursor with shared features of immaturePaneth and goblet cells. Terminal differentiation of this orother precursors into Paneth cells appears to be blockedthroughout the crypt. In contrast, the upper crypt and lowerhalf of the villus appear capable of supporting proliferation,survival, and differentiation of the intermediate cell to granulegoblet and then mature common goblet cells but not to Panethcells.

These contrasting responses of the Paneth and goblet celllineages to SV40 TAg expression may reflect the effects of themicroenvironment along the crypt-villus axis. Adopting andexpressing a Paneth cell fate may require that precursors mi-

grate to the crypt base rather than to the villus. Such a notionis consistent with a recent study in which epithelial cell migra-tion from the crypt to the villus tip was slowed in transgenicand chimeric-transgenic mice by forced expression of E-cad-herin (68). Despite the slowing of cell migration out of thecrypt, terminal differentiation markers that are normally onlyexpressed on the villus were not produced in the crypt. Thisfinding suggests that terminal differentiation of the principalsmall intestinal epithelial lineages is largely cell nonautono-mous and apparently dependent upon instructions obtained atspecific positions along the crypt-villus axis. An intriguingquestion raised by these considerations is what determineswhether a cell migrates out of a crypt or down to its base.

Paneth Cells and Intestinal Neoplasia—A Leu 3 Stop sub-stitution at codon 850 in the 2845-residue mouse Apc protein isassociated with the development of multiple intestinal neo-plasms (Min; Refs. 17 and 69). Comparable germ line muta-tions in the human APC gene also leads to multiple gut ade-nomas (familial adenomatous polyposis). Mom1 is a semi-dominant modifier of tumor multiplicity in Min/1 animalslocated on mouse chromosome 4 (70). As noted in the Introduc-tion, genetic studies indicate that Pla2g2a is a candidate genefor Mom1 (23–25). Two Pla2g2a alleles have been described.One allele contains a frameshift mutation (Mom-1S) and isencountered in C57BL/6J and 129/Sv-Pas mice. The other al-lele does not contain the mutation (Mom1R) and is found inAKR/J, MA/MyJ, BALB/cByJ, and Mus castaneus animals (25).Mom1S is associated with a 4–8-fold greater number of intes-tinal adenomas.

Current evidence indicates that a Min adenoma ariseswithin a small intestinal crypt and that the initiated cell maybe the stem cell or one of its immediate descendants (e.g. Ref.71). The mechanism by which the secreted phospholipase A2

encoded by Pla2g2a could influence initiation or progression isunclear at present. Our experiments indicate that neither amarked reduction in Pla2g2a in the crypt (CR2-tox176 mice)nor an expansion of the population of intermediate and granulegoblet cells that produce this phospholipase A2 (CR2-TAg mice)are associated with apparent changes in the properties of thecrypt stem cell or its descendants.

Although the Mom1 allele of FVB/N mice has not been char-acterized, crosses of FVB/N CR2-tox176 and their nontrans-genic littermates to Min/1 animals with Mom1S or Mom1R

alleles illustrate how Paneth cell ablation can be used as apreliminary test of the effects of gene products produced in thislineage on tumorigenesis.2 Targeting potential regulators oftumorigenesis to the apical secretory apparatus of Paneth cellsusing nucleotides 26500 to 1 34 of the mouse cryptdin-2 genecould result in their export to the stem cell zone, therebytesting their effects in Min/1 or other mouse models. Theselatter experiments illustrate how the Paneth cell can be used asa tool for delivering a variety of molecules to a critical region ofthe crypt where decisions about proliferative status and lineageallocation are made.

Ablation of the Paneth Cell Lineage Does Not Appear to AffectHost-microbial Interactions or the Spatial Organization of theDiffuse GALT—Small intestinal crypts are normally devoid ofdetectable micro-organisms. Surprisingly, our lineage ablationexperiments suggest that the anti-microbial factors producedby Paneth cells may not be required to prevent colonization ofthese crypts. Nonetheless, Paneth cells may play other roles inhost-microbial interactions. For example, the anti-microbial

2 Intepretation of such an experiment is facilitated by the results of arecent comparison of germ-free and conventionally raised C57BL/6-ApcMin/1 mice. The data indicated that the gut microflora does notstrongly affect tumor multiplicity (72).

Effects of Paneth Cell Ablation23738

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

products released from Paneth cells could function to define thecomposition and/or density of the intestinal microflora. Thisidea is consistent with the fact that bacterial density in thesmall intestine is generally 3 to 4 orders of magnitude less thanin the cecum and colon (50) which lack Paneth cells. Membersof the microflora are able to establish niches at various posi-tions along the duodenal-colonic axis even in the face of theepithelium’s continuous replacement. Colonization of this openecosystem begins soon after birth and progresses through aseries of stages resulting in complex but stable climax commu-nities predominated by Gram-negative anaerobes (29). “Au-tochthonous” members of the flora represent indigenous spe-cies that normally inhabit a given ecological niche.“Allochthonous” members are colonizers that do not normallyoccupy a given niche. Their existence in the ecosystem eitherresults from a transient “passing through” or from conditionsthat significantly disrupt the stability of the autochthonousflora (e.g. starvation or treatment with antibiotics). The com-position and flux of the autochthonous flora play a significantrole in the ability of pathogens to gain a foothold in the ecosys-tem. Cephalocaudal differences in Paneth cell cryptdin expres-sion together with the differential sensitivity of various micro-organisms to different cryptdins may help define the gut’sautochthonous (and allochthonous) flora. With these thoughtsin mind, it will be important to evaluate the effects of Panethcell ablation on the density, composition, and cephalocaudaldistribution of components of the gut’s microbiota. This can bedone using conventional mice or animals that have been raisedunder germ-free conditions and then inoculated with one ormore species of bacteria that normally reside in the gut.

CR2-tox176-mediated ablation of Paneth cells in mice thatare free of pathogens had no demonstrable effect on the crypt-villus distribution of members of the diffuse GALT. This find-ing not only provides another piece of evidence that host-mi-crobial interactions are not markedly deranged but alsoindicates that this lineage does not function, directly or indi-rectly, in establishing or maintaining the asymmetric distribu-tion of some critical components of the diffuse GALT.

While previous genetic experiments have shown that abla-tion of components of the diffuse GALT (e.g. intraepithelial gd

T-cells) affects epithelial homeostasis (57), reciprocal experi-ments designed to test the effects of ablating epithelial celllineages on GALT homeostasis have not been described. Thislikely reflects prior difficulty in defining the spatial organiza-tion of the diffuse gut-associated immune system, a problemthat can now be overcome by using tyramide signal amplifica-tion to increase the sensitivity of immunohistochemicalsurveys.

There are several notable asymmetries in the crypt-villusdistribution of components of the diffuse GALT in adult FVB/NCR2-tox176 transgenic mice and their normal littermates.First, CD81 cells are principally intraepithelial and are re-stricted to the villus, whereas CD41 cells are distributed alongthe length of the crypt-villus axis where they reside principallyin the lamina propria. Second, gd T-cells are limited to thevillus and its intraepithelial compartment. In contrast, ab T-cells are not restricted to the intraepithelial compartment andare distributed along the length of crypt-villus units. The ab-sence of CD81 and gd T-cells from the crypt may reflect, at leastin part, the lack of microbial colonization of this region of thecrypt-villus axis. If true, these cells may be useful markers tofollow if and when colonization occurs.

Our studies suggest that further assessment of the contribu-tions of Paneth cells to the regulation of host-microbial andmicrobial-immune interactions might require provocativetests. Possibilities include introduction of pathogens, with or

without members of normal flora, into germ-free CR2-tox176animals and their germ-free nontransgenic littermates orcrosses between conventionally raised CR2-tox176 animals andmice that are genetically predisposed to develop inflammatorybowel disease. An example of the latter would be interleukin-10knockout mice who develop small intestinal mucosal inflam-mation when housed in a conventional animal facility but notwhen they are raised in a specific pathogen-free state (73).

Acknowledgments—We thank David O’Donnell, Maria Karlsson,Chandra Oleksiewicz, Elvie Taylor, Bill Coleman, and Marlene Greenfor superb technical assistance. We are grateful to Bill Dove (Universityof Wisconsin, Madison) for sharing unpublished data about Min/1 mice.

REFERENCES

1. Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 537–5622. Bjerknes, M., and Cheng, H. (1981) Am. J. Anat. 160, 51–633. Bjerknes, M., and Cheng, H. (1981) Am. J. Anat. 160, 65–754. Loeffler, M., Birke, A., Winton, D., and Potten, C. (1993) J. Theor. Biol. 160,

471–4915. Potten, C. S., and Loeffler, M. (1990) Development 110, 1001–10206. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992) J. Cell Biol. 119,

493–5017. Hall, P. A., Coates, P. J., Ansari, B., and Hopwood, D. (1994) J. Cell Sci. 107,

3569–35778. Cheng, H. (1974) Am. J. Anat. 141, 481–5029. Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 461–480

10. Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 503–52011. Cheng, H. (1974) Am. J. Anat. 141, 521–53612. Cheng, H., Merzel, J., and Leblond, C. P. (1969) Am. J. Anat. 126, 507–52513. Troughton, W. D., and Trier, J. S. (1969) J. Cell Biol. 41, 251–26814. Tan, X., Hsueh, W., and Gonzalez-Crussi, F. (1993) Am. J. Path. 142,

1858–186515. de Sauvage F. J., Keshav, S., Kuang, W. J., Gillett, N., Henzel, W., and

Goeddel, D. V. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9089–909316. Poulsen, S. S., Nexo, E., Olsen, P. S., Hess, J., and Kirkegaard, P. (1986)

Histochemistry 85, 389–39417. Su, L.-K., Kinzler, K. W., Vogelstein, B., Preisinger, A. C., Moser, A. R.,

Luongo, C., Gould, K. A., and Dove, W. F. (1992) Science 256, 668–67018. Wilson, C. L., Heppner, K. J., Rudolph, L. A., and Matrisian, L. M. (1995) Mol.

Biol. Cell 6, 851–86919. Wilson, C. L., Heppner, K. J., Labosky, P. A., Hogan, B. L. M., and Matrisian,

L. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1402–140720. Mulherkar, R., Desai, S. J., Rao, R. S., Wagle, A. S., and Deo, M. G. (1991)

Histochemistry 96, 367–37021. Mulherkar, R., Rao, R. S., Wagle, A. S., Patki, V., and Deo, M. G. (1993)

Biochem. Biophys. Res. Commun. 195, 1254–126322. Harwig, S. S. L., Tan, L., Qu, X., Cho, Y., Eisenhauer, P. B., and Lehrer, R. I.

(1995) J. Clin. Invest. 95, 603–61023. MacPhee, M., Chepenik, K. P., Liddell, R. A., Nelson, K. K., Siracusa, L. D.,

and Buchberg, A. M. (1995) Cell 81, 957–96624. Gould, K. A., Dietrich, W. F., Borenstein, N., Lander, E. S., and Dove, W. F.

(1996) Genetics 144, 1769–177625. Gould, K. A., Luongo, C., Moser, A. R., McNeley, M. K., Borenstein, N.,

Shedlovsky, A., Dove, W. F., Hong, K., Dietrich, W. F., and Lander, E. S.(1996) Genetics 144, 1777–1785

26. Ghoos, Y., and VanTrappen, G. (1971) Histochem. J. 3, 175–17827. Peeters, T., and VanTrappen, G. (1975) Gut 16, 553–55828. Ouellette, A. J., and Selsted, M. E. (1996) FASEB J. 10, 1280–128929. Savage, D. C. (1977) Annu. Rev. Microbiol. 31, 107–13330. Darmoul, D., and Ouellette, A. J. (1996) Am. J. Physiol. 271, G68–G7431. Hauft, S. M., Kim, S. H., Schmidt, G. H., Pease, S., Rees, S., Harris, S., Roth,

K. A., Hansbrough, J. R., Cohn, S. M., Ahnen, D. J., Wright, N. A., Goodlad,R. A., and Gordon, J. I. (1992) J. Cell Biol. 117, 825–839

32. Bry, L., Falk, P., Huttner, K., Ouellette, A., Midtvedt, T., and Gordon, J. I.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10335–10339

33. Huttner, K. M., Selsted, M. E., and Ouellette, A. J. (1994) Genomics 19,448–453

34. Seeburg, P. H. (1982) DNA (N. Y.) 1, 239–24935. Maxwell, F., Maxwell, I. H., and Glode, L. M. (1987) Mol. Cell. Biol. 7,

1576–157936. Hogan, B., Constantini, F., and Lacy, E. (eds) (1986) Cold Spring Harbor

Laboratory Laboratory, Cold Spring Harbour, NY37. Luna, L. G. (ed) (1968) Manual of Histologic Staining Methods of the Armed

Forces Institute of Pathology, McGraw-Hill Inc., New York38. Selsted, M. E., Miller, S. I., Henschen, A. H., and Ouellette, A. J. (1992) J. Cell

Biol. 118, 929–93639. Roth, K. A., Hertz, J. W., and Gordon, J. I. (1990) J. Cell Biol. 110, 1791–180140. Bry, L., Falk, P. G., Midtvedt, T., and Gordon, J. I. (1996) Science 273,

1380–138341. Cohn, S. M., and Lieberman, M. W. (1984) J. Biol. Chem. 259, 12456–1246242. Falk, P., Roth, K. A., and Gordon, J. I. (1994) Am. J. Physiol. 266, G987–G100343. Hunyady, B., Mezey, E., Pacak, K., and Palkovits, M. (1996) Histochem. Cell

Biol. 106, 447–45644. Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980) Int. Rev. Cytol. 68,

251–30145. Simon, T. C., Roberts, L. J. J., and Gordon, J. I. (1995) Proc. Natl. Acad. Sci.

U. S. A. 92, 8685–868946. Merzel, J., and Leblond, C. P. (1969) Am. J. Anat. 124, 281–305

Effects of Paneth Cell Ablation 23739

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

47. Hertzog, A. J. (1937) Am. J. Pathol. 13, 351–36048. Castro, N. M., and Sasso, S. (1959) Lin. Acta Anat. 38, 345–35249. Breitman, M. L., Rombola, H., Maxwell, I. H., Klintworth, G. K., and Bern-

stein, A. (1990) Mol. Cell. Biol. 10, 474–47950. Gustafsson, B. E. (1982) Scand. J. Enterol. 77, 117–13151. Snel, J., Heinen, P. P., Blok, H. J., Carman, R. J., Duncan, A. J., Allen, P. C.,

and Collins, M. D. (1995) Int. J. Syst. Bacteriol. 45, 780–78252. Davis, C. P., and Savage, D. C. (1974) Infect. Immun. 10, 948–95653. Klaasen, H. L., Koopman, J. P., Van den Brink, M. E., Van Wezel, H. P., and

Beynen, A. C. (1991) Arch. Microbiol. 156, 148–15154. Umesaki, Y., Okada, Y., Matsumoto, S., Imaoka, A., and Setoyama, H. (1995)

Microbiol. Immunol. 39, 555–56255. Jepson, M. A., Clark, M. A., Simmons, N. L., and Hirst, B. H. (1993) Infect.

Immun. 61, 4001–400456. Neutra, M. R., Frey, A., and Kraehenbuhl, J.-P. (1996) Cell 86, 345–34857. Komano, H., Fujiura, Y., Kawaguchi, M., Matsumoto, S., Hashimoto, Y.,

Obana, S., Mombaerts, P., Tonegawa, S., Yamamoto, H., Itohara, S., Nanno,M., and Ishikawa, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6147–6151

58. Roberts, S. J., Smith, A. L., West, A. B., Wen, L., Findley, R. C., Owen, M. J.,and Hayday, A. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11774–11779

59. Lew, D., Brady, H., Lausing, K., Yaginuma, K., Theill, L. E., Stauber, C.,Karin, M., and Mellon, P. L. (1993) Genes Dev. 7, 683–693

60. Li, Q., Karam, S. M., and Gordon, J. I. (1995) J. Biol. Chem. 270, 15777–15788

61. Chandrasekaran, C., Coopersmith, C. M., and Gordon, J. I. (1996) J. Biol.Chem. 271, 28414–28421

62. Schmidt, G. H., Winton, D. J., and Ponder, B. A. J. (1988) Development 103,785–790

63. Winton, D. J., Blount, M. A., and Ponder, B. A. J. (1988) Nature 333, 463–46664. Wong, M. H., Hermiston, M. L., Syder, A. J., and Gordon, J. I. (1996) Proc.

Natl. Acad. Sci. U. S. A. 93, 9588–959365. Montero, C., and Erlandsen, S. L. (1978) Anat. Rec. 190, 127–14166. Calvert, R., Bordeleau, G., Grondin, G., Vezina, A., and Ferrari, J. (1988) Anat.

Rec. 220, 291–29567. Tuccari, G., and Barresi, G. (1984) Basic Appl. Histochem. 28, 177–18268. Hermiston, M. L., Wong, M. H., and Gordon, J. I. (1996) Genes Dev. 10,

985–99669. Moser, A. R., Pitot, H. C., and Dove, W. F. (1990) Science 247, 322–32470. Dietrich, W. F., Lander, E. S., Smith, J. S., Moser, A. R., Gould, K. A., Luongo,

C., Borenstein, N., and Dove, W. (1993) Cell 75, 631–63971. Shoemaker, A. R., Moser, A. R., and Dove, W. F. (1995) Cancer Res. 55,

4479–448572. Dove, W. F., Clipson, L., Gould, K. A., Luongo, C., Marshall, D. J., Moser, A. R.,

Newton, M. A., and Jacoby, R. F. (1997) Cancer Res. 57, 812–81473. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K., and Muller, W. (1993) Cell 75,

263–274

Effects of Paneth Cell Ablation23740

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Emily M. Garabedian, Lisa J. J. Roberts, M. Shane McNevin and Jeffrey I. GordonTransgenic Mice

Examining the Role of Paneth Cells in the Small Intestine by Lineage Ablation in

doi: 10.1074/jbc.272.38.237291997, 272:23729-23740.J. Biol. Chem. 

  http://www.jbc.org/content/272/38/23729Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/272/38/23729.full.html#ref-list-1

This article cites 71 references, 34 of which can be accessed free at

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from