BASIC AND TRANSLATIONAL—ALIMENTARY TRACT ADAM10Regulates Notch Function in Intestinal Stem
Cells of Mice
Yu-Hwai Tsai,1,*Kelli L.VanDussen,2,*Eric T.Sawey,4Alex W.Wade,1Chelsea Kasper,1 Sabita Rakshit,1Riha G.Bhatt,1Alex Stoeck,1Ivan Maillard,3Howard C.Crawford,5
Linda C.Samuelson,2and Peter J.Dempsey1,2
Departments of1Pediatrics and Communicable Diseases,2Molecular and Integrative Physiology,and3Life Sciences Institute, University of Michigan,Ann Arbor,Michigan;4Department of Pharmacological Sciences,Stony Brook University,Stony Brook, New York;and5Department of Cancer Biology,Mayo Clinic,Jacksonville,Florida
BACKGROUND&AIMS:A disintegrin and metalloproteinase domain-containing protein10(ADAM10)is a cell surface shed-dase that regulates physiologic processes,including Notch signaling.ADAM10is expressed in all intestinal epithelial cell types,but the requirement for ADAM10signaling in crypt ho-meostasis is not well de?ned.METHODS:We analyzed intestinal tissues from mice with constitutive(Vil-Cre;Adam10f/f mice)and conditional(Vil-CreER;Adam10f/f and Leucine-rich repeat-con-taining GPCR5[Lgr5]-CreER;Adam10f/f mice)deletion of ADAM10.We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26NICD),or mice with intestine-speci?c disruption of Notch (Rosa26DN-MAML),to examine the effects of ADAM10deletion on cell fate speci?cation and intestinal stem cell maintenance. RESULTS:Loss of ADAM10from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation,and increased secretory cell differentiation.ADAM10deletion led to the replacement of intestinal cell progenitors with2distinct,post-mitotic, secretory cell lineages:intermediate-like(Paneth/goblet)and enteroendocrine cells.Based on analysis of Rosa26NICD and Rosa26DN-MAML mice,we determined that ADAM10controls these cell fate decisions by regulating Notch signaling.Cell lineage-tracing experiments showed that ADAM10is required for survival of Lgr5tcrypt-based columnar cells.Our?ndings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche.CONCLUSIONS: ADAM10acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling.This process is required for progenitor cell lineage speci?cation and crypt-based columnar cell maintenance.
Keywords:CBC;Intestinal Epithelium;Development; Differentiation.
T he intestinal epithelium undergoes continuous renewal to preserve tissue integrity.Wnt and Notch signaling have distinct roles in controlling proliferation and cell lineage speci?cation within the crypt compartment that must be precisely integrated to maintain intestinal homeo-stasis.1Cell lineage-tracing studies have shown that Notch signaling is active in multipotent intestinal stem cells (ISCs)2–4and that Notch activation during intestinal devel-opment leads to increased proliferation and ampli?cation of intestinal progenitors.5Notch stimulation of this mitogenic response requires functional Wnt signaling.6Our previous studies demonstrated that Notch directly targets the leucine-rich repeat-containing GPCR5[Lgr5]tcrypt base columnar cell(CBC)and is required for stem cell proliferation and survival.7However,the mechanism of Notch regulation and the extent to which Notch contributes to ISC replenishment within the crypt stem cell niche has not been clearly de?ned.
Notch controls cell fate decisions of short-lived,bipotent progenitors by regulating the key transcription factor Atoh1.6Notch1and Notch2receptors and delta-like ligands (Dll)1and Dll4control these events.8,9On Notch activation, Atoh1expression is repressed in progenitors that drive differentiation into the enterocyte lineage.10Conversely,in the absence of Notch signaling,progenitors express Atoh1 and are fated into the secretory lineage.Atoh1target genes, such as G?1,Spdef,and neurogenin-3(Neurog3),are responsible for later speci?cation events in the secretory lineage.11–14Some evidence suggests that goblet and Paneth cells have a shared lineage,7but it is unclear how multi-potent secretory progenitors are allocated and give rise to the major secretory cell types.
Canonical Notch receptor signaling is controlled by sequential processing,which requires extracellular(S2) cleavage by an a-secretase,followed by intramembrane(S3) cleavage by a presenilin-dependent g-secretase,to release the Notch intracellular domain(NICD).1A disintegrin and metalloproteinase domain-containing protein10(ADAM10) was proposed to be a candidate Notch a-secretase because ADAM10à/àmice show an embryonic lethal phenotype that resembles Notch-de?cient mice.15More recent analysis of conditional ADAM10-de?cient mice and studies using transformed ADAM10à/àmouse embryonic?broblasts have shown that ADAM10is required for ligand-induced *Authors share co-?rst authorship.
Abbreviations used in this paper:ADAM10,A disintegrin and metal-loproteinase domain-containing protein10;CBCs,crypt-based columnar cells;Dll,Delta-like ligand;EGFP,enhanced green?uorescent protein;IP, intraperitoneally;ISC,intestinal stem cell;IVZ,intervillus zone;Lgr5,Leucine-rich repeat-containing GPCR5;MMP7,matrix metalloproteinase-7; Neurog3,neurogenin-3;nGFP,nuclear green?uorescent protein;NICD, Notch intracellular domain;TX,tamoxifen;YFP,yellow?uorescent protein.
?2014by the AGA Institute
0016-5085/$36.00
https://www.doczj.com/doc/822729372.html,/10.1053/j.gastro.2014.07.003
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Notch activation during development.16However,the absolute dependency of Notch signaling on ADAM10in vivo remains controversial,as other ADAMs (eg,ADAM17)and metalloproteinases (eg,matrix metalloproteinase-7[MMP7])have been implicated in Notch activation within other contexts.16–18Here,using loss-of-function studies,we show that ADAM10is required for Notch activation in the intestine and reveal that cell-autonomous ADAM10signaling is essential for cell lineage speci ?cation and in-testinal stem cell survival.Our ?ndings also suggest a competitive advantage for Notch-activated stem cells to replenish the stem cell compartment.
Methods
Mice
All animal procedures were approved by the University Committee on the Use and Care of Animals at University of Michigan.The following mouse strains were used:Adam10f ,19Villin-Cre ,20Villin-CreERT2,20Lgr5-EGFP-ires-CreERT2,21Rosa26YFP ,22Atoh1LacZ ,23Neurog3EYFP ,24Rosa26NICD-ires-nEGFP ,25and Rosa26DNMAML-GFP .26
Protocols for all procedures are provided in the Supplementary Material .
Results
ADAM10Inactivation Leads to Reduced Viability,Altered Intestinal Morphology,and Loss of Progenitor Cell Proliferation
ADAM10is abundantly expressed on the basolateral surface of all epithelial cell types in the intestine,including Lgr5tCBCs (Figure 1A and Supplementary Figure 1A ).To determine the role of ADAM10in developing and adult in-testine,we analyzed constitutive Villin-Cre;Adam10f/f (termed Vil-Cre;Adam10f/f )and tamoxifen (TX)-inducible Villin-CreERT2;Adam10f/f (termed Vil-CreER;Adam10f/f )mice.Vil-Cre;Adam10f/f pups were born at the correct Mendelian frequency and had normal body weights and intestinal length;however,no ADAM10-de ?cient pups sur-vived beyond postnatal day 1(Supplementary Figure 1B –D ,Supplementary Table 1,data not shown).Because of this perinatal lethality,we used TX-inducible Vil-CreER;-Adam10f/f mice to examine the effect of ADAM10loss in the adult intestine.Adult Vil-CreER;Adam10f/f mice treated with TX (100mg/kg intraperitoneally [IP])for 5consecutive days achieved near-complete ADAM10deletion from intes-tinal epithelium (Supplementary Figure 1E and F ).These mice rapidly lost weight,became moribund,and did not survive beyond 7–9days from the initial TX treatment.Histologic analysis of the small intestine from newborn Vil-Cre;Adam10f/f mice revealed that the epithelium was less cellular and villi were blunted with more goblet cells (Figure 1B ).Importantly,the intervillus zone (IVZ)showed a marked reduction in the number of proliferating cells,with only a few Ki67tcells located at the villus boundary (Figure 1B ).Similar morphologic and proliferative changes were observed in TX-treated adult Vil-CreER;Adam10f/f
mice,with a signi ?cant reduction in bromodeoxyuridine tcells throughout the crypt (Figure 1C ).This was associated with a marked increase in active caspase-3staining (Supplementary Figure 1F ),indicating that apoptosis accompanied the loss of cell proliferation.These results demonstrate that loss of ADAM10in either the immature or adult intestinal epithelium leads to diminished viability associated with altered intestinal morphology and reduced proliferation.
ADAM10De ?ciency Leads to Increased Secretory Cell Differentiation
Further investigation into the differentiation status of newborn small intestine from Vil-Cre;Adam10f/f mice revealed dramatic increases in secretory cell marker expression for goblet,(PAS/AB t,Muc2t),Paneth (MMP7t,lysozyme t),and enteroendocrine (chromogranin A,CHGA t)cells (Figure 2A ,data not shown).Analogous increases in secretory cell differentiation were found in TX-treated adult Vil-CreER;Adam10f/f mice,but here,an expanded crypt compartment was observed in which the mid/upper crypt regions were completely ?lled with differentiated secretory cells (Figure 2D ).Conversely,the enterocyte marker,alka-line phosphatase,was markedly reduced in both ADAM10-de ?cient models (data not shown).Morphometric and quantitative polymerase chain reaction analyses con ?rmed the dramatic increase in secretory cell differentiation observed in both ADAM10-de ?cient models (Figure 2B ,C ,E ,and F ).Together,these results indicate that ADAM10plays an important role in cell fate speci ?cation of the small in-testine and that ADAM10loss leads to increased secretory cell differentiation.
ADAM10De ?ciency Produces Distinct
Intermediate-Like (Paneth/Goblet)and Endocrine Cell Populations
In g -secretase inhibitor –treated mice,we previously showed that increased intestinal secretory cell differentia-tion is associated with the appearance of distinct intermediate-like cells that coexpress Paneth and goblet cell markers.7Similarly,in both ADAM10-de ?cient models,costaining for MMP7and MUC2detected intermediate-like cells throughout the intestine,including the colon (Figure 2and Supplementary Figure 2A ).In newborn Vil-Cre;Adam10f/f mice,the intermediate-like cells were restricted to the IVZ (Figure 2A ),and in TX-treated adult Vil-CreER;Adam10f/f mice,the intermediate-like cells were located primarily in the mid/upper crypt region.Ultra-structural analysis con ?rmed that mature Paneth cells were present at the crypt base.By contrast,the intermediate-like cells located in the mid-crypt region of ADAM10-de ?cient mice showed ultrastructural features of both Paneth and goblet cells,including mucus granules and variably sized electron-dense granules characteristic of Paneth cells (Supplementary Figure 2B ).Costaining of the endocrine-speci ?c marker CHGA with MMP7showed that the intermediate-like cells did not overlap with endocrine cells
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in either ADAM10-de ?cient model (Figure 2A and D ).Together,these data demonstrate that crypt progenitor cells are fated toward distinct intermediate-like (Paneth/goblet)or endocrine cell lineages in the absence of ADAM10signaling.
ADAM10Controls Notch Signaling Associated With Cell Fate Decisions
Notch signaling controls cell lineage speci ?cation in the intestine.In the absence of Notch signaling,the default programming of transit-amplifying progenitors is toward secretory cell differentiation.1The similarity of this intestinal phenotype with that observed in ADAM10-de ?cient mice led us to examine whether ADAM10directly regulates Notch signaling.In both ADAM10-de ?cient models,gene expression analysis showed no signi ?cant changes in Notch1,Notch2,Dll-1,and Dll-4
messenger RNA levels,suggesting that expression of Notch signaling components was not compromised (data not shown).However,quantitative polymerase chain re-action analysis revealed a 2-fold reduction in expression of Notch target genes Hes1and HeyL (Figures 3A and D and Supplementary Figure 3A ).Consistent with a loss of Notch signaling,immunohistochemical analysis con ?rmed that nuclear Hes1staining was completely absent from
the IVZ of newborn Vil-Cre;Adam10f/f
mice (Supplementary Figure 3B ).In addition,examination of Notch downstream effectors revealed a dramatic increase in the expression of key transcription factors required for secretory lineage speci ?cation,including Atoh1,G ?1,Spdef,Neurog3,and Sox9(Figure 3A and D ).To examine Atoh1and Neurog3expression at a cellular level,Atoh1lacZ and Neurog3YFP reporter mice,23,24respectively,were bred to both ADAM10-de ?cient models.Consistent with the expansion of secretory cell lineages,abundant
X-gal
Figure 1.Reduced pro-genitor cell proliferation on ADAM10deletion.(A )ADAM10staining in adult small intestine of wild-type and Lgr5-EGFP-ires-CreER mice.Right :Co-staining with GFP (red ).(B )H&E (upper)and Ki67tstaining (lower,ar-rowheads )of small intes-tine from newborn
Adam10f/f
and Vil-Cre;A-dam10f/f mice.Quanti ?-cation of Ki67tcells per ?eld (n ?4).(C)H&E (up-per )and bromodeoxyur-idine (BrdU)tstaining (lower,arrowheads )of adult small intestine from Vil-CreER;Adam10f/f mice treated with TX (100mg/kg IP)or vehicle for 5consecutive days and analyzed the next day.Quanti ?cation of BrdU tcells in crypts (n ?3).*P <.05;**P <.01;***P <.001;and ****P <.0001.Scale bars in (A )left ?200m m and right ?35m m;in (B )upper ?100m m and lower ?50m m;and in (C )?200m m (in inset ?65m m).
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Figure 2.ADAM10deletion converts intestinal crypt progenitors to a secretory cell fate.(A –C ),Newborn small intestine from Adam10f/f and Vil-Cre;Adam10f/f mice (n ?4).(D –E )Adult small intestine from Vil-CreER;Adam10f/f mice treated with TX as described in Figure 1(n ?5à7).(A ,D )Periodic acid –Schiff/Alcian Blue staining (upper )and immuno ?uorescence analysis of goblet (MUC2),Paneth (MMP7),and enteroendocrine (CHGA)cell markers (lower ).Intermediate (Paneth/goblet)and CHGA tcells indicated by arrowheads .(B ,E )Morphometric analysis of secretory cell types.(C ,F )Quantitative polymerase chain re-action analysis of cell lineage markers.*P <.05;**P <.01;***P <.001;and ****P <.0001.Scale bars in all ?100m m,except in (C )upper ?200m m.
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staining representing Atoh1tcells was observed throughout the IVZ and crypt compartment (Figure 3B and E ).Similarly,more Neurog3tcells representing early endocrine progenitors were found in newborn and adult intestine that partially colocalized with the endocrine marker,CHGA.In addition,the expanded Neurog3tcell population was distinct from MUC2t-expressing inter-mediate cells (Figure 3C and F and Supplementary Figure 3B and C ).Together,these results demonstrate that Notch and its downstream signaling events are compromised after ADAM10inactivation in both devel-oping and adult intestine.
Notch Is the Dominant Signaling Pathway Regulated by ADAM10
To verify that Notch signaling is the principal pathway controlled by ADAM10in the intestine,we tested whether overexpression of constitutively active Notch could override ADAM10de ?ciency.For this experiment,we used the Notch gain-of-function allele Rosa26NICD-ires-nGFP (termed Rosa26-NICD
),in which complementary DNAs encoding the mouse Notch1-ICD and nuclear green ?uorescent protein (nGFP)have been targeted to the Rosa26locus.25Consistent with previous reports,Vil-Cre;Rosa26NICD/tmice displayed a loss of secretory cell differentiation.5Importantly,the
compound
Figure 3.ADAM10deletion leads to a loss of Notch signaling.(A –C )Perinatal small intestine from Adam10f/f and Vil-Cre;Adam10f/f mice.(D –F )Adult small intestine from Vil-CreER;Adam10f/f mice treated with TX as described in Figure 1.(A,D )Quantitative polymerase chain reaction analysis of Notch gene targets (upper )and cell lineage-speci ?c transcription factors (n ?3à7).(B,E ),X-Gal staining of Atoh1LacZ/treporter mice.(C,F )Immunostaining of Neurog3YFP/treporter mice.Arrow-heads mark Neurog3tcells.*P <.05;**P <.01;and ***P <.001.Scale bars in (B )?100m m;in (C )?65m m;in (E )?200m m;in (F )?135m m (in insets ?65m m).
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Vil-Cre;Adam10f/f ;Rosa26NICD/tmice,despite the loss of ADAM10(data not shown),had an intestinal phenotype that was indistinguishable from Vil-Cre;Rosa26NICD/tmice,indi-cating that activated Notch could override the loss of ADAM10(Figure 4A ).Next,we examined the Notch loss-of-function allele,Rosa26DNMAML/t,in which a complemen-tary DNA encoding a GFP-tagged dominant-negative Mastermind-like construct is targeted to the Rosa26locus.26In newborn Vil-Cre;Rosa26DNMAML/tmice,co-staining of cell lineage markers revealed the appearance of intermediate-like cells within the IVZ and increased goblet and endo-crine cell populations that closely resembled the phenotype found in Vil-Cre;Adam10f/f mice (Figure 4A and Supplementary Figure 4A ).In addition,progenitor cell proliferation and nuclear Hes1immunostaining mirrored the changes in cellular differentiation observed in the different Notch mutants (Figure 4B and Supplementary Figure 4B and C ).Similar results were obtained when the same Notch mutants were examined in the adult intestine using the TX-inducible Vil-CreER line (Supplementary Figure 5).Together,these results demonstrate that Notch signaling is the dominant pathway regulated by ADAM10in the developing and adult intestine.
Mosaic ADAM10Deletion Produces Crypt Degeneration That Can Be Rescued By Activated Notch
The pronounced changes in intestinal differentiation and rapid morbidity observed on ADAM10deletion prevented
long-term fate analysis of adult ADAM10-de ?cient ISCs.To circumvent this problem,the TX treatment was modi ?ed to produce a mosaic pattern of ADAM10recombination within crypts (Supplementary Figure 6).Vil-CreER;Adam10f/f mice given a single TX dose (100mg/kg,IP)displayed reduced ADAM10deletion,but animals still lost weight,became moribund,and had to be sacri ?ced at day 9(Figure 5A ).Histologic evaluation of the small intestine revealed striking changes,with villus atrophy and crypt degeneration (Figure 5B ).All degenerating crypts were ADAM10-de ?cient,had reduced proliferation,and numerous apoptotic cells were detected within the gut lumen and in degenerating crypts (Figure 5C and data not shown).By contrast,adjacent wild-type crypts contained tall,pseudos-trati ?ed epithelial cells that were ADAM10tand highly proliferative (bromodeoxyuridine t)suggesting that these crypts were involved in an intestinal repair response (Figure 5B and C ).
Next,we tested whether Notch activation could prevent crypt degeneration by examining Vil-CreER;Adam10f/f ;Rosa26NICD/tand genotype control mice under the same experimental conditions.Strikingly,TX-treated Vil-CreER;-Adam10f/f ;Rosa26NICD/tmice were healthy,maintained weight,and had normal intestinal histology (Figure 5A and B ).Co-staining for nGFP (a surrogate marker of NICD)and ADAM10showed that NICD(nGFP)tADAM10-de ?cient crypts were readily detected and had proliferation compa-rable with wild-type controls (Figure 5C ).Similar results were obtained when recombination was induced with 4-hydroxytamoxifen (4-OH-TX)in crypt organoid
cultures
Figure 4.ADAM10-mediated Notch signaling is required for cell lineage speci ?cation and progeni-tor cell proliferation.(A )Immuno ?uorescence anal-ysis of cell lineage markers in the newborn small in-testine from Vil-Cre;
Adam10f/f
and in different Notch mutants.(B )Quan-ti ?cation of Ki67tcells per ?eld (n ?3à4).P values of group comparisons be-tween Adam10f/f and other genotypes are shown.*P <.05;**P <.01;****P <.0001.Scale bars ?100m m.
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derived from the same mice.4-OH-TX –treated organoids from Vil-CreER;Adam10f/f mice showed reduced cell prolif-eration and growth failure,and 4-OH-TX –treated organoids
from Vil-CreER;Adam10f/f ;Rosa26NICD/tmice displayed robust cell proliferation and organoid growth similar to wild-type organoids (Figure 6A ).Together,these
results
Figure 5.ADAM10loss leads to crypt degenera-tion that can be overridden by activated Notch.(A –C )Adult mice were given a single TX (100mg/kg,IP)dose and analyzed at day 9post treatment (n ?3).(A )Body weight analysis.(B )H&E analysis.Black boxes are shown at high magni ?cation (lower ).In Vil-CreER;Adam10f/f ;Rosa 26YFP/tmice,crypt degeneration (black aster-isks )is observed adjacent to crypts with increased cellularity (orange asterisk ).(C )Immuno ?uorescence analysis of ADAM10with bromodeoxyuridine (up-per )and YFP or NICD(nGFP)(lower ).In Vil-CreER;Adam10f/f ;Rosa 26YFP/tmice,degenerat-ing crypts are ADAM10-de ?cient and lack cell proliferation (white aster-isks ),and adjacent regen-erating crypts express ADAM10and are highly proliferative (orange asterisk ).In Vil-CreER;-Adam10f/f ;Rosa26NICD/tmice,NICD(nGFP)tADAM10-de ?cient crypts (white asterisks )are ?lled with NICD(nGFP)tcells (arrows )and maintain cell proliferation (arrowheads ).p values from group com-parisons of genotypes Vil-CreER;Adam10f/t;Rosa 26YFP/tvs Vil-CreER;-Adam10f/f ;Rosa26YFP/t(red )and Vil-CreER;-Adam10f/f
;Rosa26YFP/tvs Vil-CreER;Adam10f/f ;Rosa 26NICD/t(blue )are shown in (A ).*P <.05;**P <.01;***P <.001;****P <.0001.
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demonstrate that Notch activation could protect ADAM10-de ?cient crypts from degeneration and restore organoid growth,suggesting that ADAM10is required for mainte-nance of ISCs.Consistent with this interpretation was the observed loss of the Notch transcriptional target and stem cell marker Olfm4from ADAM10-de ?cient organoids (Figure 6B )and from ADAM10-de ?cient immature and adult intestine (Supplementary Figure 7).
ADAM10-Mediated Notch Signaling Is Required for Long-Term Maintenance of Lgr5tCrypt-Based Columnar Cells
To formally test the fate of ADAM10-de ?cient ISCs,lineage tracing was performed using Vil-CreER;Adam10f/f ;Rosa26YFP and genotype control mice given a single TX dose (50mg/kg,IP)and then chased over time.At day 5of
chase,
Figure 6.Activated Notch restores proliferation and Olfm4expression in ADAM10-de ?cient orga-noids.(A )Organoid cul-tures from adult mice treated with 4-hydroxytamoxifen (4-OH-TX)(5m M)for 12hours and then analyzed at day 5post treatment.Whole-mount analysis of morphology (upper )and 5-ethyl deoxyuridine (EdU)staining (mid-upper ).Co-staining of ADAM10,EdU,and YFP or NICD(nGFP)(lower panels ).Arrows indicate NICD(nGFP)tcells.Scale bars in (B )?200m M (in inset ?65m M);in (C )?65m M;in (D )all ?200m M.(B )Quantitative polymerase chain reaction analysis of ISC markers in 4-OH-TX –treated organoid cultures (n ?3).*P <.05;***P <.001;****P <.0001.
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an equivalent percentage of yellow?uorescent protein (YFP)tcrypts were found in both genotypes.In TX-treated Vil-CreER;Adam10f/f;Rosa26YFP crypts,both YFPt/ADAM10àand YFPà/ADAM10àcells were detected due to the sto-chastic nature of recombination(Supplementary Figure8A and B).However,by day28of chase,selection of mono-clonal lineage-tracing events had occurred.In TX-treated genotype control mice,ADAM10t/YFPtcrypt-villus units were detected.By contrast,in TX-treated ADAM10-de?cient mice,there was a marked reduction in the number of YFPtlineage-tracing events,and all of these had“escaped”ADAM10deletion,indicating that ADAM10-de?cient cells observed at day5had not survived(Figure7A).Similarly, no YFPtADAM10-de?cient crypt-villus units were detected when lineage tracing was performed using Lgr5-CreER;Adam10f/f;Rosa26YFP mice(Figure7C).The failure to detect any YFPtADAM10-de?cient crypt-villus units in either genetic model,together with the inability to detect DN-MAMLtcrypt-villus units when the same experiments were performed with Rosa26DNMAML allele(data not shown), indicate that ADAM10-de?cient and Notch-de?cient Lgr5tCBCs were not capable of maintaining crypt homeostasis. Notch-Activated ADAM10-De?cient Lgr5tCrypt-Based Columnar Cells Have a Competitive Advantage to Occupy the Stem Cell Niche To test whether activated Notch could rescue ADAM10-de?cient ISCs,lineage tracing was performed using Vil-CreER;Adam10f/f;Rosa26NICD/tand genotype control mice. At day5of chase,NICD(nGFP)tcells were found in both genotypes.However,the Vil-CreER;Adam10f/f;Rosa26NICD/tmice had a signi?cant increase in the percent of NICD(nGFP)tcrypts and these crypts primarily expressed NICD(nGFP)t/ADAM10àcells(Supplementary Figure8C). By day28of chase,NICD(nGFP)tADAM10-de?cient crypt-villus units were readily detected,indicating that activated Notch had rescued the ability of ADAM10-de?cient ISCs to populate the crypt compartment(Figure7B).In addition, there was a6-fold increase in the percentage of NICD(nGFP)tlineage-tracing events observed in Vil-CreER;Adam10f/f;Rosa26NICD/tmice compared with genotype controls,suggesting that there was a selective advantage in the ADAM10-de?cient state for NICD(nGFP)tADAM10-de?cient ISCs to occupy the stem cell niche (Figure7B).When this same experiment was repeated using the Lgr5-CreER line,a5-fold increase in the number of NICD(nGFP)tADAM10-de?cient lineage-tracing events was observed in Lgr5-CreER;Adam10f/f;Rosa26NICD/tmice compared with genotype controls(Figure7D).Notch signaling was clearly active in NICD(nGFP)tADAM10-de?cient crypts,as demonstrated by the loss of secretory differentiation and the restoration of crypt cell proliferation (Supplementary Figures8D and9).Interestingly,although there was no signi?cant difference in progenitor cell pro-liferation between NICD(nGFP)t/ADAM10àand NICD(nGFP)t/ADAM10tcrypts,NICD(nGFP)tADAM10-de?cient crypt villus units represented the majority (>97%)of lineage-tracing events in ADAM10-de?cient state.This increased NICD(nGFP)tlineage tracing was not due to an expansion of enhanced GFP(EGFP)t-expressing Lgr5tcrypts in the ADAM10-de?cient state,as the total number of these crypts remained constant in both geno-types(see Supplementary Methods).These results demon-strate that activated Notch can rescue ADAM10-de?cient Lgr5tCBCs.Although the precise mechanism(s)for how ADAM10-de?ciency leads to enhanced NICD(nGFP)tlineage tracing is still unclear,the results clearly demonstrate that Notch-activated ADAM10-de?cient stem cells have a competitive advantage to occupy the stem cell niche.We conclude that cell-autonomous ADAM10-mediated Notch signaling plays an important role in the maintenance of Lgr5tCBCs and crypt homeostasis.
Discussion
The progenitor cell compartment of the small intestine is comprised of slowly and actively cycling ISCs located within the crypt base.27Although the interplay between these different ISC populations and transit-amplifying pro-genitors is crucial for crypt homeostasis,the signaling pathways required for ISC function within the stem cell niche are not well de?ned.In this study,we demonstrate that cell-autonomous ADAM10activity functions within the crypt compartment to regulate the Notch signaling essential for maintenance of ISCs and progenitor cell lineage speci-?cation.We also demonstrate that Notch is the dominant substrate regulated by ADAM10within the crypt compart-ment,consistent with ADAM10de?ciency in other organ systems in vivo.19,28In addition,we show that activated Notch can rescue ADAM10-de?cient Lgr5tCBCs and enhance their ability to repopulate the crypt compartment, indicating that Notch-activated stem cells have a competi-tive advantage to occupy the stem cell niche and re-establish ISC homeostasis.
It is well-established that Notch signaling plays a crucial role in controlling cell lineage speci?cation of intestinal progenitors.1Earlier Notch loss-of-function studies showed that Notch inhibition resulted in goblet cell hyperplasia,8,9,29 and more recent reports have demonstrated robust dif-ferentiation of all secretory cell lineages.7,30,31Recently, VanDussen et al,showed that Notch inhibition with a g-secretase inhibitor induces accumulation of a distinct“in-termediate”cell population that expresses both Paneth and goblet cell markers throughout the intestine,including the colon.7Consistent with a loss of Notch signaling,morpho-logic and transcriptional analysis of the ADAM10-de?cient intestine showed robust secretory cell differentiation in the immature and adult intestine.In addition,analysis of Notch loss-of-function(Rosa26DN-MAML)and gain-of-function (Rosa26NICD)mutants provided direct genetic evidence that cell-autonomous ADAM10activity regulates Notch signaling in these cell fate decisions.
Currently,the mechanism(s)by which secretory pro-genitors give rise to the major secretory cell lineages remain controversial.In one model,it has been proposed that multipotent secretory progenitors must rapidly commit to a speci?c secretory cell fate,32and recent analysis of different
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Figure 7.ADAM10is required for maintenance of adult Lgr5tCBCs.(A,B )Lineage tracing of Vil-CreER;Adam10f/f ;Rosa26YFP/t,Vil-CreER;Adam10f/f ;Rosa26NICD/tand genotype control mice given a single TX (50mg/kg IP)dose and then chased for 28days (n ?3).Costaining of ADAM10and YFP or NICD(nGFP).(A )In Vil-CreER;Adam10f/f ;Rosa26YFP/tmice,no ADAM10-de ?cient cells were detected.Arrowheads indicate individual YFP tPaneth cells (Supplementary Figure 8A ).(B )In both genotypes,NICD(nGFP)tcells are detected (arrowheads ).Asterisks mark NICD(nGFP)tADAM10-de ?cient crypt-villus units.(C,D )Lineage tracing of Lgr5-CreER;Adam10f/f ;Rosa26YFP/t,Lgr5-CreER;Adam10f/f ;Rosa26NICD/tand genotype con-trol mice given a single TX (400mg/kg orogastric)dose and then chased for 28days (n ?3).Immuno ?uorescence analysis of ADAM10and YFP or NICD(nGFP).Note that EGFP t-Lgr5cells can co-express YFP or NICD(nGFP).(C )In Lgr5-CreER;Adam10f/f ;Rosa26YFP/tmice,no ADAM10-de ?cient cells were detected.Strong YFP expression from the Rosa26YFP allele masks the weaker EGFP t-Lgr5cells.(D )NICD(nGFP)tcells (arrows ),EGFP t-Lgr5cells displaying stronger cytoplasmic staining (arrowheads )and NICD(nGFP)tADAM10-de ?cient crypt-villus unit (asterisk )are shown.(A –D )Lower panels :Quan-ti ?cation of each lineage-tracing event.*P <.05;**P <.01;***P <.001;****P <.0001;na,not applicable.Scale bars ?100m m.
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Atoh1-,Neurog3-,and G?1-de?cient mouse models suggests that multipotent Atoh1tprogenitors give rise to committed enteroendocrine progenitor and bipotent goblet/Paneth progenitor populations.10–13In the ADAM10-de?cient crypts,the progenitor cell population was completely replaced by Atoh1tcells,which is indicative of cells un-dergoing secretory differentiation.In addition,we con?rmed using the Neurog3YFP reporter line that the“in-termediate”(Paneth/goblet)cells were distinct from Neu-rog3tprogenitors required for enteroendocrine lineage speci?cation.A similar accumulation of distinct intermedi-ate and endocrine cell populations was observed on expression of DN-MAML,demonstrating that cell-autonomous Notch inhibition was directly responsible for the appearance of these different secretory cell types.
Notch plays an essential role in crypt cell prolifera-tion,5,29but it is only recently that lineage-tracing and Notch inhibition studies have demonstrated that active Notch signaling is present2–4,33and required for mainte-nance of ISCs.7,34Notch inhibition led to reduced prolif-eration,loss of the stem cell marker,Olfm4,and apoptosis of CBCs,indicating that active Notch signaling is required for survival of Lgr5tCBCs.In the immature and adult intestine,intestine-speci?c ADAM10inactivation closely recapitulated these?ndings,including reduced progenitor proliferation and loss of Olfm4.Additional analysis of ADAM10-de?cient crypts in vivo or cultured organoids in vitro revealed severe crypt degeneration and organoid failure associated with a loss of progenitor proliferation and increased apoptosis.In addition,our data from orga-noid experiments clearly demonstrate that ADAM10acts in a cell autonomous manner and does not require any mesenchymal signaling to ful?ll its functions.Importantly, the ability of activated Notch to completely restore pro-genitor proliferation and cell viability in both of these settings demonstrates that ADAM10-mediated Notch signaling is essential for long-term crypt survival.Inter-estingly,a recent study using mice constitutively expressing dominant-negative ADAM10under the cryptdin-2promoter reported mislocalized Paneth cells,a phenotype linked to a loss of Eph signaling.35By contrast, our results clearly demonstrate that ADAM10de?ciency in the stem/progenitor cell compartment produces a domi-nant Notch loss-of-function phenotype.Although ADAM10 likely regulates other substrates in the crypt compart-ment,the loss of stem/progenitor cell proliferation and rapid crypt degeneration observed in our current ADAM10-de?cient models has not permitted analysis of ADAM10signaling in postmitotic intestinal cells,including Paneth cells,and this will be an important area for addi-tional investigation.
Mosaic recombination epigenetically found in the Lgr5-EGFP-ires-CreERT2line or produced by reduced TX dosing in the Villin-CreERT2line allowed long-term line-age tracing of ADAM10-de?cient ISCs.In both models, ADAM10-de?cient ISCs failed to populate the crypt compartment and no dormant ADAM10-de?cient ISCs were detected,indicating that ADAM10-de?cient ISCs did not survive long term.Similar results were found when lineage tracing was performed with the Notch loss-of-function mutant DN-MAML.The demise of ADAM10-de?cient ISCs was clearly illustrated by the ability of activated Notch to rescue ADAM10-de?cient ISCs and restore their capacity to populate the crypt-villus axis. Together,these results demonstrate that ADAM10-mediated Notch signaling is crucial for the maintenance of these ISC populations.
It has been proposed that neutral drift and symmetric cell divisions are critical elements that control ISC homeo-stasis.ISCs within the same crypt form an equipotent pop-ulation in which the loss of one stem cell can be replaced stochastically by another stem cell.36,37In addition,lineage tracing after injury or ablation of Lgr5tCBCs demonstrates that the Lgr5tstem cell compartment can be repopulated through mobilization of quiescent stem cell populations38,39 or by de-differentiation of crypt progenitors.40,41Analogous to these Lgr5tcell injury/ablation studies,our data suggest that ADAM10deletion within Lgr5tCBCs leads to stem cell loss and an imbalance within the stem cell niche that pro-motes permissive signals for wild-type ISCs and/or pro-genitor populations to re-establish ISC homeostasis. Unexpectedly,lineage tracing in the presence of activated Notch revealed a5-to6-fold increase in the total number of NICDtlineage-tracing events within the ADAM10-de?cient state compared with genotype control mice.Our data sug-gest that loss of ADAM10-de?cient CBCs creates permissive conditions in which Notch-activated stem cells have a competitive advantage to replenish the ISC compartment.A possible explanation based on the kinetics of ADAM10 deletion and functional NICD expression is that the imme-diate progeny of NICDt/ADAM10-de?cient Lgr5tCBCs ef?ciently repopulate the stem cell compartment.This result is consistent with the plasticity of secretory progenitors to revert to a stem-cell state.40,41It will be interesting to test whether this enhanced compensatory mechanism observed with active Notch signaling can occur in other regenerative processes in which imbalances of ISC homeostasis are found.
In summary,Notch is the dominant substrate regulated by ADAM10in the crypt compartment.ADAM10-mediated Notch signaling is essential for cell lineage speci?cation and maintenance of Lgr5tCBCs.Importantly,loss of ADAM10-de?cient Lgr5tCBCs created a competitive advantage for Notch-activated stem cells to repopulate the stem cell niche and restore ISC homeostasis. Supplementary Material
Note:To access the supplementary material accompanying this article,visit the online version of Gastroenterology at https://www.doczj.com/doc/822729372.html,,and at https://www.doczj.com/doc/822729372.html,/10.1053/ j.gastro.2014.07.003.
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Received February28,2014.Accepted July10,2014.
Reprint requests
Address requests for reprints to:Peter J.Dempsey,PhD,Department of Pediatrics,University of Colorado Medical School,RC26113,12700E19th Avenue,Aurora,CO80045.e-mail:peter.dempsey@https://www.doczj.com/doc/822729372.html,;fax:303-724-6538.
Acknowledgements
We thank the Michigan Gut Group for helpful discussions,Dr Sudo for Hes1 antibody,Dr Gradwohl for Neurog3YFP line,Dr Robine for Villin-Cre and Villin-CreER lines and Rebecca Tucker and Connie Brindley for technical assistance.
Kelli L.VanDussen’s current af?liation is Department of Pathology and Immunology,Washington University School of Medicine,St Louis,MO.Eric T.Sawey’s current af?liation is Cold Spring Harbor Laboratory,Cold Spring Harbor,NY.Alex W.Wade’s current af?liation is Department of Biology, Johns Hopkins University,Baltimore,MD.Riha G.Bhatt’s current af?liation is Department of Pediatrics,University of Pittsburgh Medical Center, Pittsburgh,PA.Alex Stoeck’s current af?liation is Merck Research Laboratories,Boston,MA.
Con?icts of interest
The authors disclose no con?icts.
Funding
This work was supported by grants from the UM Comprehensive Cancer Center(PJD),Crohn’s and Colitis Foundation of American(PJD),and the National Institutes of Health(NIH)R01-DK093697(PJD),R01-AI091627(IM), R01-DK096972(LCS),and R01-CA159222(HCC).This work utilized the Morphology and Image Analysis Core of the Michigan Diabetes Research Center funded by NIH National Institute of Diabetes and Digestive and Kidney Diseases P60DK020572.KLV was supported by an American Gastroenterological Association Foundation Graduate Student Fellowship and a Rackham Predoctoral Fellowship.
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Supplementary Methods
Other Mouse Studies
All animal procedures were approved by the University Committee on the Use and Care of Animals at the University of Michigan.For CreERT2lines,adult(>6-week-old)mice were given TX by IP injection(25–100mg/kg)or oral gavage(400mg/kg).TX(Sigma,St Louis,MO)was sus-pended in corn oil:EtOH(95:5,v/v)at a concentration of10 mg/mL.Corn oil was used as vehicle control.For TX-induced recombination using the Vil-CreER and Lgr5-CreER lines,additional genotype controls were used, including TX-treated Adam10f/f,Vil-CreER;Adam10t/t, Lgr5-CreER;Adam10t/t,Vil-CreER;Adam10f/t,and Lgr5-CreER;Adam10f/tmice.In all cases,the intestinal pheno-type of all TX-treated genotype controls appeared normal.
The Rosa26LacZ/tstrain1was used to measure TX-induced recombination ef?ciency in adult mice (Supplementary Figure6).Brie?y,Vil-CreER;Adam10f/f; Rosa26LacZ/tmice were given a single TX(25,50,or100 mg/kg,IP)dose and analyzed over time.X-gal staining was performed at day6after TX treatment and the percentages of X-galtcrypts were determined from histologic analysis.
For analysis of Vil-Cre;Adam10f/f and Adam10f/f during development,E12.5to E18.5embryos from timed-pregnant dams were dissected on ice in phosphate-buffered saline (PBS).Excised gastrointestinal tracts were?xed in4% paraformaldehyde(PFA)in PBS for4hours,transferred to 30%sucrose overnight at4 C,and then OCT embedded. Bromodeoxyuridine Administration
and Tissue Collection
Mice were injected IP with50mg/kg bromodeoxyur-idine(BrdU;Roche,5mg/mL in0.9%saline)2hours before tissue collection.For histologic analysis,intestine tissue was either?xed in cold4%PFA in PBS for4hours,transferred to30%sucrose overnight at4 C and OCT embedded,or ?xed in4%PFA overnight and then paraf?n-embedded.For RNA and protein analysis,full-thickness newborn intestine or scraped mucosa from adult intestine was collected. Histologic Analysis
Frozen and paraf?n(3–5m m)sections were used for histologic analysis and immunohistochemistry,as described previously.2Primary antibodies included rat anti-ADAM10 (MAB946,1/400;R&D,Minneapolis,MN),rabbit anti-chromogranin A(15160,1/500;Abcam,Cambridge,MA), rat anti-MMP7(1/200;Nashville,TN),rabbit anti-lysozyme (A0099,1/800;Dako,Carpinteria,CA),rabbit anti-Mucin2 (sc-15334,1/500;Santa Cruz Biotechnology,Santa Cruz, CA),rabbit anti-Neurog3(F25A1B3,1/4000;Developmental Studies Hybridoma Bank,Iowa City,IA),rat anti-Hes1 (kindly provided by T.Sudo,Toray Industries,1/50),rab-bit anti-Ki67(Novocastra,NCL-Ki67P,1/200),rabbit anti-active caspase3(9664,1/400;Cell Signaling,Danvers MA),rabbit anti–E-cadherin(9587,1/100;Cell Signaling), rabbit anti–b-catenin(3195,1/100;Cell Signaling),rabbit anti–smooth muscle actin–Cy3conjugated(C6198,1/500;Sigma),rat anti-BrdU(OBT0030G,1/400;Accurate Chemi-cal,Westbury,NY),and rabbit-anti synaptophysin(18-0130, 1/100,Carlsbad,CA).Rat anti-Hes1antibody was detected using anti-rat antibody conjugated to horseradish peroxi-dase.Neurog3antibody binding was detected as described previously.3All other primary antibodies were detected using appropriate secondary antibodies conjugated to?uo-rescein isothiocyanate or Cy3.Fluorescein and rhodamine-labeled Dolichos Bi?orus Agglutinin lectin were obtained from Vector Labs(Burlingame,CA;FL-1031,1/1000;FL-1032,1/1000).40,6-diamidino-2-phenylindole,alkaline phosphatase(Vector,SK-5100),periodic acid-Schiff,and Alcian blue staining were performed using manufacturer’s protocol.Microscopy was performed with an Olympus IX-71 microscope equipped with a DP72digital camera or with an Olympus FluoView500confocal microscope.
To clearly visualize the loss of ADAM10staining in ADAM10-de?cient crypts particularly within lineage-tracing experiments,images have been pseudocolored.In most cases,ADAM10staining has been pseudocolored green,and GFP antibody staining for YFP(Rosa26YFP allele,cytoplasmic YFP),GFP(Lgr5-EGFP-ires-CreERT2allele;cytoplasmic GFP),GFP(Rosa26DNMAML-GFP allele,cytoplasmic GFP), YFP(Neurog3EYFP allele,cytoplasmic YFP),and GFP (Rosa26NICD-ires-nEGFP,nuclear GFP)were pseudocolored red.
Whole-mount b-galactosidase(b-galt)staining of Rosa26LacZ and Atoh1LacZ intestines was carried out as described previously.4Brie?y,intestines were?xed for2 hours in0.2%glutaraldehyde,2%PFA in PBS,rinsed,and then incubated in X-gal staining buffer(5mM K3Fe(CN)6,5 mM K4Fe(CN)63H2O,2mM MgCl2,0.02%NP40,0.1% deoxycholate,1mg/mL X-gal in PBS overnight at room temperature in the dark.Intestines were then embedded in OCT,sectioned,and X-galtstaining visualized by microscopy.
For electron microscopy,jejunum was excised(n?3/ group),emersion?xed in2.5%glutaraldehyde in0.1M Sorensen’s buffer(pH7.4)overnight at4 C,post?xed for1 hour in1%OsO4,dehydrated,and Epon embedded.Ultra-thin sections were stained with uranyl acetate and lead citrate and then examined using a Philips CM100electron microscope.Images were recorded digitally using a Hama-matsu ORCA-HR digital camera system.
Lineage Tracing of Crypt-Villus Units
For analysis of lineage tracing,a crypt-villus unit was de?ned on histologic cross-section when the crypt lumen is contiguous with the crypt base and both sides of the crypt exit onto the villus.A single lineage-tracing event was de?ned as a crypt-villus unit in which labeled cells populate the entire crypt-villus axis,including ISCs,crypt TA cells, and villus epithelium.To count crypt-villus units,the entire small intestine was?xed with4%PFA in PBS,transferred to 30%sucrose overnight and then“Swiss”rolled before OCT embedding.Frozen sections(3–5m m)were cut,immuno-stained for the appropriate markers and lineage-tracing events were counted from the entire length of the small intestine(at least n?3mice/group).
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For Vil-CreERT2lineage tracing,the number of YFPtor NICD(nGFP)tcrypt-villus units were counted from immu-nostained frozen sections of small intestine and expressed as a percentage of total crypts counted(n?3per genotype). For day-5lineage tracing,the total number of crypts counted for each genotype was Vil-CreER;Adam10f/t; Rosa26YFP/t:814,889,929;Vil-CreER;Adam10f/f; Rosa26YFP/t:1091,1075,1277;Vil-CreER;Adam10f/t; Rosa26NICD/t:1929,2104,2047;Vil-CreER;Adam10f/f; Rosa26NICD/t:2347,2058,2114).For day-28lineage tracing,the total number of crypts counted for each geno-type was:Vil-CreER;Adam10f/t;Rosa26YFP/t:1296,979, 995;Vil-CreER;Adam10f/f;Rosa26YFP/t:1167,8771102; Vil-CreER;Adam10f/t;Rosa26NICD/t:2096,2218,2076;Vil-CreER;Adam10f/f;Rosa26NICD/t:2301,2418,2730).
For lineage-tracing experiments in which the Lgr5-EGFP-ires-CreERT2and Rosa26YFP alleles were used in combina-tion,anti-GFP antibody staining was used to detect both EGFP and YFP.YFP expression was much stronger and masked EGFP expression in Lgr5tcells.For lineage-tracing experiments in which the Lgr5-EGFP-ires-CreERT2and Rosa26NICD-ires-nGFP alleles were used in combination,anti-GFP antibody was used to detect both EGFP and nuclear GFP(which is a surrogate marker of NICD expression).The nGFP expression is termed NICD(nGFP)tin the text.The cytoplasmic EGFP expression from the Lgr5-EGFP-ires-CreERT2allele was much stronger and masked the NICD(nGFP)texpression from the Rosa26NICD-ires-nGFP allele in Lgr5tcells.However,lineage tracing could be performed with the Rosa26NICD-ires-nGFP allele and was used for the identi?cation of ADAM10-de?cient NICD(nGFP)t/Lgr5tcrypt-villus units.
For Lgr5-EGFP-ires-CreERT2lineage tracing,the number of YFPtor NICD(nGFP)tcrypt-villus units was counted from immunostained frozen sections of full-length small intestine and expressed as the total number of YFPtor NICD(nGFP)tcrypt-villus units per intestine.In addition, the total number of Lgr5t/EGFPtcrypts per small intestine for each genotype was counted(n?3à4per genotype). For day-28lineage tracing,the total number of EGFPt-expressing Lgr5crypts counted for each genotype was Lgr5-CreER;Adam10f/t;Rosa26YFP/t:1701,1302,1559; Lgr5-CreER;Adam10f/f;Rosa26YFP/t:1647,1681,1955;Lgr5-CreER;Adam10f/t;Rosa26NICD/t:788,1340,1062;Lgr5-CreER;Adam10f/f;Rosa26NICD/t:1651,1567,1034,856). Positional Scoring of Bromodeoxyuridinet
Cells in Crypts
Cell positional distribution of BrdUtcells in crypts was determined as described previously.5Brie?y,frozen sec-tions(3–5m m)of small intestine were cut,immunostained with BrdU antibody,and then nuclei were stained with40,6-diamidino-2-phenylindole.Well-orientated half-crypts(!15 crypts per intestine,n?3mice/group)were scored for the presence of BrdUtnuclei at all of the cell positions along the crypt axis,with position1being the middle of the crypt base.The frequency of each event at each position was plotted.Morphometric Analysis of Secretory
Cell Lineages
Sequential frozen sections(3–5m m)were cut from P0 and adult small intestine and then immunostained with speci?c secretory cell lineage markers de?ned as follows: endocrine cells,CHGAtonly;Paneth cells,MMP7tonly; goblet cells,MUC2tonly;and intermediate(Paneth/goblet) cells,MMP7t/MUC2tdouble-positive.The total number cells positive for each cell marker were counted from40?images obtained using an Olympus IX-71microscope equipped with a DP72digital camera.For P0intestine,the percent of each secretory cell type(ie,total number of positive cells for each secretory lineage marker/total number of epithelial cells?100)was determined from the cross-sectional images.For adult intestine,the total number of each secretory cell type per crypt(number of positive cells for each cell lineage marker/total number of crypts) was determined(n!10crypts per adult intestine,n?3 mice/group).
Organoid Culture
Mouse organoid cultures isolated from jejunal crypts were established and maintained as described previously.6 The basic culture medium(advanced Dulbecco’s modi?ed Eagle medium/F12supplemented with1?penicillin/ streptomycin,10mmol/L HEPES,1?Glutamax,1?B27,1?N2(all from Life Technologies,Carlsbad,CA),and1mmol/L N-acetylcysteine(Sigma)was supplemented with40ng/mL murine recombinant epidermal growth factor(PeproTech, Rocky Hill,NJ),25ng/mL murine recombinant Noggin (PeproTech),R-spondin2(conditioned medium,5%?nal volume),and Wnt-3A(conditioned medium,5%?nal vol-ume).Conditioned media were produced using HEK293T cells stably transfected with mouse R-spondin2-Fc7(pro-vided by Drs Whitsett and Shroyer,University of Cincinnati, OH)and L cells stably transfected with mouse Wnt-3A (ATCC,Manassas,VA).To induce gene deletion,4-hydroxytamoxifen(5m mol/L;Sigma)was added to the culture medium for24hours and then cultured for up to5 days.Frozen(3–5m m)sections were used for histologic analysis and immunohistochemistry prepared from orga-noids?xed in4%PFA and embedded in https://www.doczj.com/doc/822729372.html,anoid cell proliferation was assessed using the Click-iT EdU Imaging Kit(Invitrogen).Brie?y,organoids grown in Matrigel were labeled in vitro with10m mol/L5-ethyl deoxyuridine solu-tion for1hour at37 C in regular culture https://www.doczj.com/doc/822729372.html,anoids were then?xed with4%PFA for15minutes and then permeabilized with0.5%Triton X-100in PBS prior to detection with the Click-iT reaction and Alexa Fluor488 using manufacturer’s protocol.Whole-mount imaging of5-ethyl deoxyuridine–labeled organoids was performed with an Olympus IX-71microscope equipped with a DP72digital camera.For RNA analysis,organoid cultures for each treatment group were collected in triplicate.
Gene Expression Analysis
Gene expression was determined by quantitative reverse transcription polymerase chain reaction with n?3à7mice
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per group.RNA was isolated from intestinal tissue or organoids using Trizol(Invitrogen)followed by puri?cation with the RNeasy kit(Qiagen,Valencia,CA).Reverse tran-
scription reactions used1m g RNA and SuperScript III kits (1808-051;Invitrogen)as recommended by the manufac-turer.Quantitative reverse transcription polymerase chain reaction was performed using an ABI7000Cycler with SYBR Green Master Mix(A&B Applied Biosystems,Carlsbad, CA;4367659)and primer sets listed in Supplementary Table2.Each20m L reaction contained1m L reverse-transcribed product and250nM of each primer.Gene expression levels were determined in triplicate and normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase,which remained the same in the various groups.
Western Blot Analysis
Full-thickness newborn small intestine and scraped adult mucosa were collected and lysed in RIPA buffer con-taining protease and phosphatase inhibitor cocktails(Roche, Nutley,NJ).Cell lysates(50m g)were mixed with Laemmli sample buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis for Western blot anal-ysis using antibodies against rat–anti-mouse ADAM10 (MAB946,1/200;R&D System)and mouse anti–b-actin (A-1978,1/3000;Sigma).
Statistical Analysis
Quantitative data are presented as mean±SEM and analyzed by2-tailed Student’s t-test.Differences between groups were determined by1-way or2-way analysis of
Supplementary References
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2.VanDussen KL,Samuelson LC.Mouse atonal homolog1
directs intestinal progenitors to secretory cell rather than absorptive cell fate.Dev Biol2010;346:215–223.
3.Lopez-Diaz L,Jain RN,Keeley TM,et al.Intestinal Neu-
rogenin3directs differentiation of a bipotential secretory progenitor to endocrine cell rather than goblet cell fate.
Dev Biol2007;309:298–305.
4.Shroyer NF,Helmrath MA,Wang VY,et al.Intestine-
speci?c ablation of mouse atonal homolog1(Math1) reveals a role in cellular homeostasis.Gastroenterology 2007;132:2478–2488.
5.Potten CS,O’Shea JA,Farrell CL,et al.The effects of
repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine.Cell Growth Differ2001;
12:265–275.
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cells build crypt-villus structures in vitro without a mesenchymal niche.Nature2009;459:262–265.
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Authors names in bold designate shared co-?rst authors.
variance using Tukey’s post-hoc method of multiple com-parisons.P<.05was considered signi?cant.
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Supplementary Figure1.Analysis of ADAM10deletion in the developing and adult small intestine.(A)Immuno?uorescence analysis of ADAM10expression in e13.5,e16.6,and P0small intestine(top panel)and co-staining of ADAM10with b-catenin, CHGA,MUC2,lysozyme,smooth muscle actin(SMA),and synaptophysin(SYN)in the adult small intestine(lower panels). Arrowheads indicate ADAM10coexpression with speci?c cell markers or cell types.(B–D)Analysis of ADAM10recombination in the newborn small intestine of Vil-Cre;Adam10f/f mice.(B)Quantitative polymerase chain reaction analysis of ADAM expression in messenger RNA isolated from full-thickness newborn small intestine.**P<.01.(C)Immuno?uorescence staining of ADAM10in Vil-Cre;Adam10f/f and genotype control mice.Arrowheads show retention of ADAM10expression in cells of the lamina propria and smooth muscle.(D)Western blot analysis of ADAM10expression in cell lysates isolated from full-thickness small intestine.(E,F)Analysis of ADAM10recombination in the adult small intestine of Vil-CreER;Adam10f/f mice treated with TX as described in Figure1.(E)Quantitative polymerase chain reaction analysis of ADAM expression in messenger RNA isolated from scraped small intestine.***P<.001.(F)Immuno?uorescence staining of ADAM10(upper panel).Arrowheads show retention of ADAM10expression in cells of the lamina propria and smooth muscle.ADAM10co-staining with activated caspase-3(white arrowheads)in vehicle-and TX-treated Vil-CreER;Adam10f/f mice(middle panel).Quanti?cation of caspase-3tcells per total crypt epithelial cells(lower panel).***P<.001.Scale bars in(A)upper?65m m;middle and lower?all35m m, except SMA and SYN?200m m;in(C)?100m m(in inset?65m m);in(F)upper?200m m,and lower?65m m.
Supplementary Figure 2.Intermediate (Paneth/goblet)cells are detected in the adult small intestine and colon on ADAM10loss.(A,B )Vil-CreER;Adam10f/f mice treated with TX as described in Figure 1.(A )Immuno ?uorescence analysis of secretory cell lineage markers in the colon.White boxes are shown at high magni ?cation in adjacent panels.Upper panel :Intermediate (Paneth/goblet)cells expressing MMP7and MUC2are marked with white arrowheads .Lower panel :White arrowheads indicate increased numbers of distinct enteroendocrine (CHGA t)cells found in the ADAM10-de ?cient colon.(B )Electron microscope analysis showing mature Paneth cells at the crypt base of the small intestine in both vehicle-and TX-treated mice (yellow asterisks ).However,in TX-treated mice,cells in the mid/upper crypt region express ultrastructural features of both Paneth and goblet cells (red arrowheads ).Inset is shown at higher magni ?cation (right ).Scale bars in (A )?135m m (in inset ?65m m);in (B )?10m m,right panel ?3.3m m.
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Supplementary Figure 3.Analysis of Notch targets and neurogenin 3expression upon ADAM10deletion.(A )Quantitative polymerase chain reaction analysis of Hes and Hey gene expression from the small intestine.Left panel :Newborn Vil-Cre;Adam10f/f (n ?3à4).Right panel :Adult Vil-CreER;Adam10f/f mice treated with TX as described in Figure 1(n ?6à7).*P <.05.Messenger RNA expression pro ?ling was performed on full-thickness P0intestine or adult mucosal scrapings,which likely explains the modest reductions in Hes/Hey gene expression compared with dramatic changes in downstream epithelial cell-speci ?c gene targets.(B )Hes1and Neurog3immunostaining in newborn small intestine.Upper panel :In Vil-Cre;Adam10f/f mice,there is a loss of nuclear Hes1expression (black arrowheads )within the IVZ (hatched line ).Black asterisks indicate nonspeci ?c cytoplasmic staining of goblet cells.Lower panel :White arrowheads indicate Neurog3tcells.(C )Left panel :Neurog3(YFP)staining in the newborn small intestine from Vil-Cre;Adam10f/f ;Neurog3YFP/tand Adam10f/f ;Neurog3YFP/tre-porter mice.Increased numbers of Neurog3t(YFP t)cells (white arrowheads )are found primarily in the IVZ.Right panel :Co-staining of Neurog3(YFP)and CHGA in the adult small intestine from Vil-CreER;Adam10f/f ;Neurog3YFP/treporter mice treated with TX as described in Figure 1.Increased numbers of Neurog3t(YFP t)(white arrowheads )and CHGA tcells that partially colocalize with each other are observed in TX-treated mice.Scale bars in (B )?100m m;in (C )left ?100m m and right ?135m m (in inset ?65m m).
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