Molecular Properties of Adult Mouse Gastric and Intestinal Epithelial Progenitors in Their Niches
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m512118200
ISSN1083-351X
AutoresMarios Giannakis, Thaddeus S. Stappenbeck, Jason C. Mills, Douglas G. Leip, Michael Lovett, Sandra W. Clifton, Joseph E. Ippolito, Jarret Glasscock, Manimozhiyan Arumugam, Michael R. Brent, Jeffrey I. Gordon,
Tópico(s)Digestive system and related health
ResumoWe have sequenced 36,641 expressed sequence tags from laser capture microdissected adult mouse gastric and small intestinal epithelial progenitors, obtaining 4031 and 3324 unique transcripts, respectively. Using Gene Ontology (GO) terms, each data set was compared with cDNA libraries from intact adult stomach and small intestine. Genes in GO categories enriched in progenitors were filtered against genes in GO categories represented in hematopoietic, neural, and embryonic stem cell transcriptomes and mapped onto transcription factor networks, plus canonical signal transduction and metabolic pathways. Wnt/β-catenin, phosphoinositide-3/Akt kinase, insulin-like growth factor-1, vascular endothelial growth factor, integrin, and γ-aminobutyric acid receptor signaling cascades, plus glycerolipid, fatty acid, and amino acid metabolic pathways are among those prominently represented in adult gut progenitors. The results reveal shared as well as distinctive features of adult gut stem cells when compared with other stem cell populations. We have sequenced 36,641 expressed sequence tags from laser capture microdissected adult mouse gastric and small intestinal epithelial progenitors, obtaining 4031 and 3324 unique transcripts, respectively. Using Gene Ontology (GO) terms, each data set was compared with cDNA libraries from intact adult stomach and small intestine. Genes in GO categories enriched in progenitors were filtered against genes in GO categories represented in hematopoietic, neural, and embryonic stem cell transcriptomes and mapped onto transcription factor networks, plus canonical signal transduction and metabolic pathways. Wnt/β-catenin, phosphoinositide-3/Akt kinase, insulin-like growth factor-1, vascular endothelial growth factor, integrin, and γ-aminobutyric acid receptor signaling cascades, plus glycerolipid, fatty acid, and amino acid metabolic pathways are among those prominently represented in adult gut progenitors. The results reveal shared as well as distinctive features of adult gut stem cells when compared with other stem cell populations. The adult gastrointestinal tracts of humans and mice contain large populations of multipotential stem cells that produce an impressive number of descendant epithelial cells each day: ∼70 billion in humans (∼0.25 kg) and 200 million in mice (1Croft D.N. Cotton P.B. Digestion. 1973; 8: 144-160Crossref PubMed Scopus (44) Google Scholar, 2Potten C.S. Cancer Metastasis Rev. 1992; 11: 179-195Crossref PubMed Scopus (298) Google Scholar). The morphologic features of adult mouse gut epithelial lineage progenitors have been characterized using tritiated thymidine labeling and electron microscopic autoradiography (3Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 461-479Crossref PubMed Scopus (545) Google Scholar, 4Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 259-279Crossref PubMed Scopus (323) Google Scholar). In the stomach, the stem cell niche is positioned in the mid-portion (isthmus) of tubular mucosal invaginations known as gastric units, whereas small intestinal and colonic stem cells reside at or near the base of crypts of Lieberkühn (4Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 259-279Crossref PubMed Scopus (323) Google Scholar, 5Bjerknes M. Cheng H. Am. J. Anat. 1981; 160: 77-91Crossref PubMed Scopus (166) Google Scholar). Analyses of genetic mosaic mice indicate that adult gastric units and intestinal crypts are monoclonal; all epithelial cells in each of these anatomically distinct structures are apparently derived from a single ancestor occupying the highest position in the stem cell hierarchy (6Bjerknes M. Cheng H. Gastroenterology. 1999; 116: 7-14Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 7Bjerknes M. Cheng H. Am. J. Physiol. 2002; 283: G767-G777Crossref PubMed Scopus (136) Google Scholar, 8Wong M.H. Saam J.R. Stappenbeck T.S. Rexer C.H. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12601-12606Crossref PubMed Scopus (113) Google Scholar). Knock-out mice have provided direct evidence that the Wnt pathway is important for maintaining a dividing small intestinal stem cell population, and that Notch signaling is involved in specification of its descendant secretory cell lineages (9Radtke F. Clevers H. Science. 2005; 307: 1904-1909Crossref PubMed Scopus (585) Google Scholar, 10van Es J.H. van Gijn M.E. Riccio O. van den Born M. Vooijs M. Begthel H. Cozijnsen M. Robine S. Winton D.J. Radtke F. Clevers H. Nature. 2005; 435: 959-963Crossref PubMed Scopus (1268) Google Scholar, 11Fre S. Huyghe M. Mourikis P. Robine S. Louvard D. Artavanis-Tsakonas S. Nature. 2005; 435: 964-968Crossref PubMed Scopus (721) Google Scholar). Nonetheless, we know relatively little about the signaling and metabolic pathways that are active in adult gastric and intestinal stem cells within their niches, the degree to which these progenitors share properties with one another, and how they compare with other characterized adult or embryonic stem cell populations. To address this issue, we have sequenced cDNA libraries prepared from laser capture microdissected adult gastric and small intestinal epithelial progenitors (GEPs 3The abbreviations used are: GEP, gastric epithelial progenitor; EST, expressed sequence tag; GO, Gene Ontology; SiEP, small intestinal epithelial progenitor; Atp4b, noncatalytic β-subunit of H+/K+-ATPase; Defcr2, cryptdin-2; DSS, dextran sodium sulfate; LCM, laser capture microdissection; IPA, ingenuity pathways analysis; BrdUrd, bromodeoxyuridine; Dcamkl1, double cortin and calcium/calmodulin-dependent protein kinase-like-1; Mapk14, mitogen-activated protein kinase 14; CgA, chromogranin A; GSII, Griffonia simplificolia II; HSC, hematopoietic stem cell; ESC, embryonic stem cell; NSC, neural stem cell; GABAA, γ-aminobutyric acid, type A; Vegf, vascular endothelial growth factor; PBS, phosphate-buffered saline; Ddc, dopa decarboxylase; Aoc3, amine oxidase, copper containing 3; Aldh9a1, aldehyde dehydrogenase 9 family, member A1; Gad1, glutamic acid decarboxylase 1; IPA, Ingenunity Pathways Analysis. ; SiEPs), taking advantage of two gnotobiotic transgenic mouse models where these cells are represented in increased numbers in discrete regions of gastric units and crypts of Lieberkühn. The resulting data sets were analyzed using a combination of GO functional annotations and a variety of software tools that allow analysis of the statistical significance of GO term enrichments and of the observed representation of components of canonical signaling and metabolic pathways. Animals—Germ-free FVB/N mice that express an attenuated diphtheria toxin A fragment (tox176) under the control of nucleotides -1,035 to +24 of mouse Atp4b (noncatalytic β-subunit of H+/K+-ATPase; Ref. 12Mills J.C. Andersson N. Hong C.V. Stappenbeck T.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14819-14824Crossref PubMed Scopus (90) Google Scholar) or nucleotides -6500 to +34 of mouse Defcr2 (cryptdin-2; Ref. 13Stappenbeck T.S. Mills J.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1004-1009Crossref PubMed Scopus (123) Google Scholar) were maintained in plastic gnotobiotic isolators (14Hooper L.V. Mills J.C. Roth K.A. Stappenbeck T.S. Wong M.H. Gordon J.I. Methods Microbiol. 2002; 31: 559-589Crossref Google Scholar) under a strict 12-h light cycle. Animals were given an autoclaved chow diet (B&KUniversal, East Yorkshire, UK) ad libitum. Wild-type C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were maintained free of specified pathogens in micro-isolator cages within a barrier facility and fed the same autoclaved chow as their germ-free counterparts. These conventionally raised C57BL/6J mice (10 weeks old) were given a 2.5% aqueous solution of dextran sodium sulfate (DSS; TDB Consultancy AB, Uppsala, Sweden) for 7 days as drinking water to induce a regenerative epithelial response in colonic crypts (15Pull S.L. Doherty J.M. Mills J.C. Gordon J.I. Stappenbeck T.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 99-104Crossref PubMed Scopus (491) Google Scholar). All experiments involving animals were performed using protocols approved by the Washington University Animal Studies Committee. Laser Capture Microdissection (LCM)—General methods for preparing the mouse gut for cryosectioning and LCM are described in Ref. 16Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 168-196Google Scholar. Specific methods for navigated LCM of GEPs from the expanded isthmal domain of gastric units in the corpus region of the stomachs of 14-week-old male germ-free Atpb4-tox176 mice are described in Ref. 12Mills J.C. Andersson N. Hong C.V. Stappenbeck T.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14819-14824Crossref PubMed Scopus (90) Google Scholar. Protocols for LCM of SiEPs from the base of crypts located at the junction between the middle and distal thirds of the small intestines of 4-week-old Defcr2-tox176 mice are provided in Ref. 13Stappenbeck T.S. Mills J.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1004-1009Crossref PubMed Scopus (123) Google Scholar. Epithelial cells were also retrieved by LCM from the lower thirds of crypts located in the descending colons of DSS-treated C57Bl/6 mice (15Pull S.L. Doherty J.M. Mills J.C. Gordon J.I. Stappenbeck T.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 99-104Crossref PubMed Scopus (491) Google Scholar). Cells were harvested using a PixCell IIe LCM System and CapSure HS LCM caps (Arcturus Bioscience). Total cellular RNA was extracted (10,000 cells/LCM population; PicoPure RNA isolation kit; Arcturus Bioscience), and its quality was evaluated using an Agilent 2100 bioanalyzer. Construction of cDNA Libraries—Detailed protocols and explanatory figures with the sequences of primers used for cDNA synthesis and library construction are available from the authors and in Ref. 17Hawkins R.D. Bashiardes S. Helms C.A. Hu L. Saccone N.L. Warchol M.E. Lovett M. Hum. Mol. Genet. 2003; 12: 1261-1272Crossref PubMed Scopus (49) Google Scholar. cDNA was synthesized from 5 ng of RNA on oligo(dT)-linked paramagnetic beads (Dynal Norway) in the presence of a modified "SMART" oligonucleotide (Clontech). Oligonucleotide dimers were removed by digestion with NotI. Bead-linked cDNA was amplified by PCR (four cycles) using the SMART primer and a hybrid primer consisting of oligo(dT) linked to an external and more specific priming site. The original bead-linked cDNAs were captured magnetically and stored. Amplified cDNAs in the supernatant were subsequently amplified for an additional seven cycles of PCR using the more specific external priming sites. cDNA products were passed through a Sephadex G-50 column (Amersham Biosciences). A final round of PCR amplification (five cycles) was performed after adding ligation-independent linkers compatible with the uracil deglycosylase system (Invitrogen). The final cDNA products were size-selected (SizeSep 400 Spun Column; Amersham Biosciences) to isolate fragments >300 bp. 10-20 ng of this cDNA were used in a standard annealing mix with the UDG pAMP1 vector (Invitrogen). Aliquots of the ligation-free annealing mix were transformed directly into Escherichia coli DH10B. The GEP, SiEP, and colonic crypt transit amplifying cell libraries each contained >5 × 105 independent clones with mean insert lengths of 0.7 kb. Alignments of ESTs to the Mouse Genome—ESTs were aligned to the mm5 assembly of the mouse genome from University of California Santa Cruz (UCSC) with BLAT (gfClient version 27 × 1 obtained from UCSC) using minScore = 50 (minimum score, BLAT default scoring scheme) and minIdentity = 95 (minimum percent identity). The alignments were then filtered using minScore = 50 (on a match = 1, mismatch = -1, and gapOpen = -4 scoring scheme), minMatch = 50 (minimum number of matching bases), and minAli = 200 (minimum ratio of query in parts per thousand). The top scoring alignment for each EST was chosen from this filtered set of alignments, along with any alignment of the EST that scored within 5% of the top scoring alignment. These alignments were subsequently filtered to include only those that contained introns. Gene symbols obtained from the EST alignments to mm5 and consensus gene clustering from Unigene can be found online at genome.wustl.edu/GSCGAP/. Similar data sets were obtained when transcripts from the GEP and SiEP libraries were aligned to mm5 using BLASTN (18Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar). A subsequent, more refined alignment with EST_GENOME (19Mott R. Comput. Appl. Biosci. 1997; 13: 477-478PubMed Google Scholar) provided confident intron-exon boundaries. Transcripts that crossed splice boundaries were annotated as likely intron retention events; each group of consistent splice patterns was put into a given cluster, and each cluster, along with its individual members and their library association, was annotated. Ingenuity Pathways Analysis (IPA)—We analyzed our data sets using the following procedures. Once logged onto the IPA system, we clicked on "File" menu, then "New," and then "Analysis." We then clicked "Upload" and selected the data set to be analyzed. For the analysis of canonical signaling and metabolic pathways active in GEPs and SiEPs, the input was the Entrez Gene identifiers of an EST library (e.g. GEPs) with the genes in a data set of interest (e.g. the uniquely GO-enriched genes expressed in GEPs) designated as focus genes. Because we had EST rather than DNA microarray data, we set the expression value to zero for all genes. We clicked on "Create Analysis" followed by "Run Analysis." When the analysis was completed by IPA software, we clicked on "Canonical Pathways" and "Customize Chart," and under "Select Categories to Display" we chose both "Metabolic Pathways" and "Signaling Pathways." Under "Select Sort Order" we ranked by "Significance" and completed the procedure by clicking "Apply." For the transcriptional networks in GEPs and SiEPs, the input was the transcription-related GO-enriched GEP or SiEP Entrez Gene identifiers. When the analysis was completed by IPA software, we clicked on "Networks," selected the top two scoring networks, and clicked "View Networks." The results presented in this study are based on IPA Knowledge Base content statistics from May, 2005. Multilabel Immunohistochemistry—Sections (7 μm thick) were prepared from paraffin-embedded, Bouin's fixed stomach (corpus region) and zinc-formalin fixed small intestine harvested from normal, 12-week-old conventionally raised FVB/N mice and/or comparably aged germ-free Atpb4-tox176 animals. Mice received an intraperitoneal injection of bromodeoxyuridine (BrdUrd) 1.5 h prior to sacrifice. Sections were sequentially passed through xylene (three washes; 5 min each), 100% isopropyl alcohol (three washes; 5 min each), and deionized water (5 min). Antigen retrieval was performed by boiling slides in 10 mm citrate buffer (pH 6.0) for 10 min, followed by cooling to 23 °C, three washes in PBS (5 min each), and incubation in blocking buffer (1% bovine serum albumin, 0.3% Triton X-100 in PBS; 30 min). Sections were subsequently incubated overnight at 4 °C with the following primary antibodies and/or biotinylated lectins: (i) rabbit anti-mouse double cortin and calcium/calmodulin-dependent protein kinase-like-1 (Dcamkl1; final dilution in blocking buffer = 1:200; a gift from Christopher Walsh, Harvard Medical School); (ii) rabbit anti-mouse mitogen-activated protein kinase 14 (Mapk14/p38; 1:200; Santa Cruz Biotechnology); (iii) goat anti-BrdUrd (1:500; see Refs. 12Mills J.C. Andersson N. Hong C.V. Stappenbeck T.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14819-14824Crossref PubMed Scopus (90) Google Scholar and 13Stappenbeck T.S. Mills J.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1004-1009Crossref PubMed Scopus (123) Google Scholar); (iv) rabbit anti-bovine chromogranin A (CgA; 1:10; ImmunoStar; conjugated to fluorophore with the Zenon tricolor rabbit IgG labeling kit from Molecular Probes); (v) Griffonia simplificolia II (GSII) lectin (1:200, specific for neck cells in the FVB/N inbred strain); and (vi) Anguilla anguilla agglutinin (1:200, specific for pit cells). After an overnight incubation at 4 °C, sections were washed with PBS (three cycles; 5 min each), and antigen-antibody complexes and bound biotinylated lectins were detected with Alexa Fluor-, cyanine (Cy)3-, or fluorescein isothio-cyanate-conjugated secondary antibodies or streptavidin (Molecular Probes and Jackson ImmunoResearch). Sections were washed in PBS, incubated with bisbenzamide (Hoechst 33258; 1 μg/ml) to stain nuclei, and then examined and photographed using a Zeiss Axiovert 200M microscope. Sequencing cDNA Libraries from Laser Capture Microdissected Gut Epithelial Progenitors—GEPs, which include cells with the EM morphologic features of the presumptive multipotential stem cell and its descendant oligo-potential pre-parietal, pre-pit, and pre-neck cell line-age progenitors, were retrieved by LCM of cryosections prepared from the middle portion (corpus) of the stomachs of germ-free transgenic mice with an engineered, attenuated diphtheria toxin A fragment (tox176)-mediated ablation of their parietal cells (12Mills J.C. Andersson N. Hong C.V. Stappenbeck T.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14819-14824Crossref PubMed Scopus (90) Google Scholar, 20Syder A.J. Guruge J.L. Li Q. Oleksiewicz C. Lorenz R.G. Karam S.M. Falk P.G. Gordon J.I. Mol. Cell. 1999; 3: 263-274Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (n = three 16-week-old animals; ∼10 cells microdissected per isthmus; total of ∼2,000 LCM gastric units). Acid-producing parietal cells terminally differentiate within the isthmal stem cell niche before they migrate to the upper (pit) and lower (base) domains of the gastric unit (21Karam S.M. Anat. Rec. 1993; 236: 314-332Crossref PubMed Scopus (169) Google Scholar). tox176-mediated parietal cell ablation results in augmented GEP proliferation, with a progressive increase in their fractional representation to ∼10% of all epithelial cells in gastric units by 16 weeks of age, as judged by transmission EM (22Syder A.J. Oh J.D. Guruge J.L. O'Donnell D. Karlsson M. Mills J.C. Björkholm B.M. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3467-3472Crossref PubMed Scopus (52) Google Scholar). Germ-free Atpb4-tox176 mice were used because loss of the acid-barrier to colonization produces bacterial overgrowth in the stomach and gastritis (20Syder A.J. Guruge J.L. Li Q. Oleksiewicz C. Lorenz R.G. Karam S.M. Falk P.G. Gordon J.I. Mol. Cell. 1999; 3: 263-274Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 22Syder A.J. Oh J.D. Guruge J.L. O'Donnell D. Karlsson M. Mills J.C. Björkholm B.M. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3467-3472Crossref PubMed Scopus (52) Google Scholar). SiEPs were recovered from germ-free Defcr2-tox176 transgenic mice with a Paneth cell ablation (13Stappenbeck T.S. Mills J.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1004-1009Crossref PubMed Scopus (123) Google Scholar). This epithelial lineage is a key component of the innate immune system of the small intestine and is the only one descended from the multipotential stem cell that completes its differentiation program at the crypt base (23Cheng H. Am. J. Anat. 1974; 141: 521-535Crossref PubMed Scopus (159) Google Scholar). tox176-directed ablation of Paneth cells results in a consolidation of SiEPs at the crypt base without affecting their proliferative activity, or the differentiation programs of the three other small intestinal epithelial lineages (13Stappenbeck T.S. Mills J.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1004-1009Crossref PubMed Scopus (123) Google Scholar). The five most basal crypt epithelial cells in Defcr2-tox176 mice were harvested by LCM of cryosections prepared from the junction between the middle and distal thirds of the small intestine (n = three Defcr2-tox176 mice; total of ∼5,000 LCM crypts). A generally applicable method was employed to generate GEP and SiEP cDNA libraries starting with 5 ng of total cellular RNA from each LCM progenitor population. This method minimizes skewing of the relative abundance of expressed transcripts. Alignments of 36,641 sequenced ESTs from the two libraries to Unigene and the mm5 build of the mouse genome yielded a data set of 4031 genes expressed in GEPs and 3324 genes in SiEPs (see supplemental Table S1; a complete list is available at genome.wustl.edu/GSCGAP/ see supplemental Table S2 for a list of 52 identified alternatively spliced variants). Identification of "Biological Process" GO Terms Enriched in GEPs and SiEPs Relative to All Gastric and Small Intestinal Cells—We used GO terms and GoSurfer (24Zhong Z. Storch K.F. Lipan O. Kao M.C. Weitz C.J. Wong W.H. Appl. Bioinformatics. 2004; 3: 261-264Crossref PubMed Scopus (87) Google Scholar) to compare each adult gut epithelial progenitor data set with previously sequenced cDNA libraries generated from the intact stomachs and small intestines of conventionally raised, normal adult mice (Fig. 1). The two stomach libraries contained a total of 3303 unique Unigene clusters, whereas the two small intestinal libraries contained 2359 Unigene clusters (supplemental Table S3). GoSurfer takes one or two gene lists as input, finds GO terms associated with the genes, and visualizes them as one of three hierarchical trees corresponding to the broad GO categories of "biological process," "molecular function," and "cellular component." GoSurfer can also compare two gene lists to identify GO terms enriched to a statistically significant degree in one data set versus the other (e.g. supplemental Fig. S1). The following analysis focused on GO terms and pathways that were more prominent in progenitor cells compared with non-stem cell populations. Sixty five biological process GO terms, representing 1767 genes (transcripts), were enriched in the GEP data base over the intact adult stomach cDNA library data set (q value cutoff of 0.1). These 1767 genes, subsumed under enriched GO terms, were termed "GO-enriched." An analogous GoSurfer comparison of the SiEP data base with the intact adult mouse small intestine cDNA libraries yielded 62 GO terms, representing 1824 genes (q value cutoff of 0.1) (see step 1 in Fig. 1 plus supplemental Tables S4 and S5). The 65 GEP-enriched and 62 SiEP-enriched GO terms are listed in supplemental Tables S6 and S7, respectively, and include the biological processes of cell cycle, DNA replication, cell proliferation, morphogenesis, pattern specification, and ubiquitination. A number of transcription-related GO terms were enriched in the two gut epithelial progenitor populations (see supplemental Tables S8 and S9 for these GO terms and their associated genes, including, for example, Notch 1-4. Note that when molecular function rather than biological process GO terms were used for GoSurfer comparisons, 96% of the transcription-related molecular function GO-enriched genes were identical to the transcription-related biological process GO-enriched genes listed in supplemental Tables S8 and S9). The genes representing these transcription-related GO terms in GEPs and SiEPs were placed onto transcription factor networks using the IPA software tool. This tool utilizes a knowledge base of over one million known functional relationships among proteins. One of the two top scoring networks in GEPs is depicted in Fig. 2A and includes factors involved in Wnt/β-catenin, phosphoinositide 3-kinase/Akt kinase (PI3K/Akt), transforming growth factor-β (Tgf-β), and insulin-like growth factor-1 (Igf-1) signaling. Similarly, one of the two top scoring SiEP-associated networks is shown in Fig. 2B and includes factors that participate in JAK/Stat (Janus kinase-signal transducer and activator of transcription signaling) and cell cycle (G1/S checkpoint) regulation (see supplemental Figs. S2A and S2B for the other top scoring networks in GEPs and SiEPs). Identification of GO-enriched Genes Whose Expression Is Unique to Gut Epithelial Progenitors or Shared with Other Stem Cell Populations—We performed a second step to distinguish GO-enriched genes unique to GEPs or SiEPs from those shared with other non-gut stem cell populations. This analysis employed all published sequenced cDNA libraries of >1000 ESTs from mouse hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), and neural stem cells (NSCs). For HSCs, we combined seven libraries totaling 5018 Unigene clusters with 4191 genes resulting from our alignment to mm5 of a hematopoietic EST data set, available from the Stem Cell Data Base (see SCDb; a joint project of the labs of I. R. Lemischka, K. A. Moore, and C. Stoeckert). For the ESC data set, we incorporated three cDNA libraries with 5259 unique Unigene clusters. For NSCs we used two libraries with 5093 Unigene clusters (supplemental Table S3). Genes producing transcripts in HSCs, ESCs, and NSCs were compared with the adult whole mouse stomach libraries and with the whole small intestinal libraries using GoSurfer. The results disclosed that 243 of the 1767 GEP-associated GO-enriched genes identified from Fig. 1, step 1, were common to the other stem cell populations, whereas 574 were GO-enriched only in GEPs. Among the 1842 SiEP-associated GO-enriched expressed genes, 268 were common, whereas 742 were uniquely GO-enriched in the transcriptomes of this LCM population (Fig. 1, step 3, and supplemental Tables S10-S13). The Venn diagrams in Fig. 1 summarize the results of further comparisons of the groups of filtered GO-enriched commonly and uniquely expressed genes identified in GEPs and SiEPs, including a 253-member group shared by GEPs and SiEPs but not with the other stem cell populations, and a group of 134 shared by GEPs, SiEPs, HSCs, NSCs, and ESCs (see supplemental Tables S14-S19 for complete lists of these genes). To test the validity of this GO enrichment-filtering approach, we analyzed a control population of colonic crypt epithelial cells harvested by LCM from adult conventionally raised C57Bl/6J mice treated for 7 days with DSS in their drinking water. DSS produces a stereotyped pattern of ulcer formation in the distal colonic epithelium and a regenerative proliferative response in crypts surrounding these ulcers (15Pull S.L. Doherty J.M. Mills J.C. Gordon J.I. Stappenbeck T.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 99-104Crossref PubMed Scopus (491) Google Scholar). EM studies disclosed that the regenerating crypt cells had features of a transit-amplifying population of immature members of the enterocytic and goblet cell lineages (15Pull S.L. Doherty J.M. Mills J.C. Gordon J.I. Stappenbeck T.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 99-104Crossref PubMed Scopus (491) Google Scholar). A cDNA library was prepared from these LCM cells and sequenced, yielding 12,013 ESTs, representing 2394 unique transcripts (supplemental Table S1). No biological process GO terms were enriched in this set of gene products compared with two adult colonic cDNA libraries with 7470 Unigene clusters (supplemental Table S3). These findings indicate that the GEP and SiEP preparations obtained by LCM of gnotobiotic Atp4b-tox176 and Defcr2-tox176 mouse guts are enriched for adult gut stem cells. Signaling and Metabolic Pathways Active in GEPs and/or SiEPs—We used the IPA tool to further characterize functional properties of these gut epithelial progenitor populations. IPA annotations take into account GO annotations but are nonetheless distinct and based on a knowledge base of protein-protein interactions, extracted from the published literature. The IPA output includes signaling and metabolic pathways and a statistical assessment of their significance. This assessment utilizes a right-tailed Fisher's Exact Test to calculate the probability that genes participate in a given pathway relative to their occurrence in all other pathway annotations. Table 1 lists GEP- and SiEP-associated canonical signaling or meta-bolic pathways. The table is organized based on the groups defined by the Venn diagrams in Fig. 1 and provides a link to the corresponding pathway maps in the supplemental material. These pathway maps contain filtered GO-enriched gene products as well as all other pathway gene products that are present, albeit not enriched, in the gut progenitor EST libraries. For example, our IPA-based analysis indicated that GEPs have a statistically significant representation of Wnt/β-catenin signaling components (Table 1). Fig. 3 illustrates how GEPs and SiEPs, as well as the other stem cell populations, contain different identified combinations of GO-enriched gene products that map to the Wnt/β-catenin pathway. This provides one measure of the shared and distinctive features of these populations.TABLE 1Canonical signaling and metabolic pathways, defined by IPA, represented in GEPs and SIEPsCanonical signaling/metabolic pathwaysPathway mapsp valueEnriched genes in the pathwayA. GEPsG-protein-coupled receptor signalingFig. S3A8.90E-5aThe representation of a pathway in these progenitor populations is statistically significant (p < 0.05) based on a right-tailed Fisher's Exact Test.Drd2, Grm8, Htr2c, P2ry4, Adra1a, Htr7, Oprd1, Adcy2, Hrh1, Htr4Wnt/β-catenin signalingFig. S3B1.53E-3aThe representation of a pathway in these progenitor populations is statistically significant (p < 0.05) based on a right-tailed Fisher's Exact Test.Tle1, Dvl3, Frzb, Wnt16, Wnt9a, Acvr1, Tcf1Tyrosine metabolismFig. S3C2.25E-3aThe representation of a pathway in these progenitor populations is statistically significant (p < 0.05) based on a right-tailed Fisher's Exact Test.Aoc3, Fah, TyrSerotonin receptor signalingFig. S3D5.02E-3aThe representation
Referência(s)