Mice and Calorie restriction
Mice were housed in the Unit for Laboratory Animal Medicine at the Whitehead Institute for Biomedical Research. Lgr5-EGFP-IRES-CreERT2
mice (Strain name: B6.129P2-Lgr5tm1(cre/ERT2)Cle/J, Stock Number: 008875) were purchased from Jackson Laboratories. UbiquitinC-CreERT2
mice were obtained from the Jackson Laboratory (Strain Name: B6;129S-Tg(UBC-cre/ERT2)1Ejb/J, Stock Number: 007001). Rictor
floxed mice were generated as described in29
and backcrossed to C57BL/6 for at least 6 generations. TSC1loxp/loxp
mice were the generous gift of D. Kwiatkowski (Harvard Medical School) and backcrossed to C57BL/6 for at least 6 generations. Rosa26-CreERT2
mice (Rosa26 or R26) were obtained from the Jackson Laboratory (Strain Name: B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj
/J, Stock Number 008463). CR was achieved by providing individual or paired mice a daily portion of a chow diet fortified with vitamins and minerals amounting to 60% of the daily food intake of their ad libitum counterparts20
. All adult mice used in CR experiments were between the ages of 10 to 24 weeks and were sacrificed prior to their daily feeding. Rictor
was excised by treatment with tamoxifen suspended in corn or sunflower seed oil (Spectrum) at a concentration of 10 mg/mL, and 200 μl per 25 g of body weight was injected intraperitoneally into mice once daily for 5–7 days. Control animals received an equal volume of the tamoxifen suspension, but did not express the CreERT2 fusion protein. Mice were allowed to recover for at least 7 days after the last tamoxifen injection prior to any experiments. In vivo
fate mapping in Lgr5-EGFP-IRES-CreERT2;Rosa26loxpstoploxp-LacZ
compound mice was done with a single injection of tamoxifen given at 200 μl per 25 g. As described previously30
, rapamycin (LC Laboratories) was administered by intraperitoneal injection for 7 to 28 consecutive doses at 4 mg/kg. It was reconstituted in absolute ethanol at 10 mg/ml and diluted in 5% Tween-80 (Sigma) and 5% PEG-400 (Hampton Research) before injection. The final volume of all injections was 200 μl. Regular insulin (Lilly) was administered at 0.75 U/kg diluted in PBS 20 minutes prior to sacrificing fasted mice.
Generation of Rheb2 Transgenic mouse
transgenic mouse was produced as described before in28
. Briefly, mouse embryonic stem cells (KH2) were obtained containing a neomycin resistance gene as well as a hygromycin resistance gene lacking a promoter and an ATG start codon at the ColA1
locus. The presence of frt sites flanking the neomycin and hygromycin resistant genes allows site-specific integration of the transgene at the ColA1
locus. These embryonic cells also contain an M2rtTA transactivator at the endogenous Rosa26
promoter, which, in the presence of doxycycline, leads to the transactivation of TetO-promoter driven transgenes. The human Rheb2
) coding sequence was cloned into a vector downstream of a TetO promoter that also contained a PKG-ATG-frt element necessary for frt-site integration. This vector, along with another vector contained the FLPe recombinase, were then electroporated into the KH2 cells. As a result, the coding sequence for hsRheb2
along with a PGK promoter and the ATG initiation codon necessary for the expression of the hygromycin resistance gene is integrated into the genomic DNA of the KH2 cells at the ColA1
locus. Cells with properly integrated hsRheb2
were then selected by hygromycin resistance and subsequently injected into blastocysts. Chimeric mice were then mated with C57BL/6J mice until germline transmission of the transgene was achieved.
PCR genotyping of the hsRheb2 transgenic mouse was performed with the following primers: Rheb-tg_F: CCAATTTGTGGAAGGCGAGTT, Rheb2-TG_R: CCATGGCCTTCATGTAGCTT
Tissues were fixed in 10% formalin, paraffin embedded, and sectioned. Antigen retrieval was performed with Borg Decloaker RTU solution (Biocare Medical) in a pressurized Decloaking Chamber (Biocare Medical) for 3 minutes. Antibodies: rat anti-BrdU (1:2000; Abcam 6326), rabbit phospho-S6 Ser235/236 (1:500, CST 4858), rabbit cleaved caspase 3 (1:500; CST 9664), rabbit chromogranin A (1:3000, Abcam 15160), rabbit Lysozyme (1:2000; Thermo), rabbit Bst-1 (1:1000, Abcam 74301) and mouse Bst-1 (1:100 to 500, BD Pharmingen). For mouse Bst-1, the M.O.M (mouse on mouse) kit was used according to the manufacturer's instructions (Vector labs PK-2200). Biotin conjugated secondary donkey anti-rabbit or rat antibodies were used from Jackson ImmunoResearch. The Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs PK-6101) followed by Dako Liquid DAB+ Substrate (Dako) was used for visualization. For immunofluorescence Alexa Fluor 568 secondary antibodies (Invitrogen) were used. All antibody incubations were performed with Common Antibody Diluent (Biogenex)
In situ hybridization
The in situ probes used in this study correspond to expressed sequence tags or fully sequenced cDNAs obtained from Open Biosystems. The accession numbers (IMAGE mouse cDNA clone in parenthesis) for these probes are as follow: mouse OLFM4
BC141127 (9055739), mouse cryptdin4
BC134360 (40134597). To ensure the specificity of the probes, we generated both sense and antisense probes by in vitro transcription using DIG RNA labelling mix (Roche) according to the manufacturer's instructions and to previously published detailed methods21,31
Radiation and clonogenic microcolony assay
AL and CR adult mice were exposed to 15 Gy of ionizing irradiation from a 137-Cesium source (GammaCell) and sacrificed after 72 hours. The number of surviving crypts per length of the intestine was enumerated from H&E-stained sections.
Antibodies: rabbit anti phospho-T389 S6K1, phospho-S240/244 S6, S6K1, and S6 from CST; rabbit Bst-1 (ab74301) from Abcam; mouse anti □-actin (clone AC-15) from Sigma. Crypts or tissue were rinsed once with ice-cold PBS and lysed in ice-cold lysis buffer (50 mM HEPES [pH 7.4], 40 mM NaCl, 2 mM EDTA, 1.5 mM sodium orthovanadate, 50 mM NaF, 10 mM pyrophosphate, 10 mM glycerophosphate, and 1% Triton X-100, and one tablet of EDTA-free protease inhibitors [Roche] per 25 ml). The soluble fractions of cell lysates were isolated by centrifugation at 13,000 rpm for 10 min. Proteins extracts were denatured by the addition of sample buffer, boiled for 5 min, resolved by SDS-PAGE, and analyzed by immunoblotting.
Flow cytometry and isolation of ISCs and Paneth cells
Small intestines were removed and the fat/mesentery was dissected away. The intestinal lumen was washed with ice cold PBS (Mg-/Ca-) using a 18G feeding needle (Roboz FN-7905) until the intestines appeared white/pink. They were then opened longitudinally. The mucous was removed by gently rubbing the intestine between fingers in cold PBS. The intestines were cut into 3 to 5 mm fragments and placed into 50 ml conical tubes that were fill with ice cold 30 ml of PBS (Mg-/Ca-)/EDTA (10 mM). The samples were incubated and shook intermittently on ice for 30 minutes continuously discarding and replacing (at least 3 times) the supernatant. The fragments were then continually resuspended with ice cold 30ml PBS (Mg-/Ca-)/EDTA (10 mM) and intermittently shook on ice for 10 minutes, discarding the supernatant again for 3 times. The fragments were resuspended again with ice cold 30 ml PBS (Mg-/Ca-)/EDTA (10 mM) and incubated and intermittently shook while waiting on ice for 20 to 40 minutes. The samples were then triturated with a 10 ml pipette 1 to 2 times, and the contents were filtered twice through a 70-μm mesh (BD Falcon) into a 50 ml conical tube to remove villous material and tissue fragments. At this point the suspension was mainly composed of crypts. Crypts were removed from this step for crypt culture experiments and embeded in matrigel with crypt culture media. For ISC isolation, the crypt suspensions were centrifuged for 5 minutes at 250 g (4C or room temperature). The pellets were gently resuspended in 1.0 ml of undiluted TrypLE Express (Invitrogen) + 120 μl of DNase I (10U/μl, Roche) and transferred to 15 ml conical tubes. The samples were incubated in a 32° C water bath for 1.25 to 2 minutes, were not titurated, and were then placed on ice. 12 ml of cold SMEM was added to each sample and were gently triturated twice. The samples were then centrifuged for 5 minutes at 250 g. The pellets were resuspended and incubated for 15 minutes on ice in 0.5 to 1 ml SMEM that contained an antibody cocktail consisting of CD45-PE (eBioscience, 30-F11), CD31-PE (Biolegend, Mec13.3), Ter119-PE (Biolegend, Ter119), CD24-Pacific Blue (Biolegend, M1/69) and EPCAM-APC (eBioscience, G8.8). 12 ml of SMEM were added and the samples were centrifuged for 5 minutes at 250 g. The pellets were resuspended with 0.5–2 ml (depending on the size of the pellet) of SMEM/ 7-AAD solution (1:500 dilution). The samples were filtered through a 40-μm mesh (BD Falcon) prior to cell sorting. ISCs were isolated as Lgr5-EGFPhiEpcam+CD24low/−CD31−Ter119−CD45−7-AAD−, EGFPlow progenitors were isolated as EGFPlowEpcam+CD24low/−CD31−Ter119−CD45−7-AAD−, and Paneth cells were isolated as CD24hiSideScatterhiLgr5-EGFP−Epcam+CD31−Ter119-CD45−7-AAD− with a BD FACS Aria II SORP cell sorter into supplemented crypt culture medium. Dead cells were excluded from the analysis with the viability dye 7-AAD. When indicated, populations were cytospun (Thermo Cytospin 4) at 800 rpm for 2min, or allowed to settle at 37°C in fully humidified chambers containing 6% CO2 onto poly-L-lysine coated slides (Polysciences). The cells were subsequently fixed in 4% paraformaldehyde (pH=7.4, Electron Microscopy Sciences) prior to staining.
Crypt culture media
Isolated crypts were counted and embeded in matrigel (BD Bioscience 356231 growth factor reduced) that contains 1 μM Jagged (Ana-Spec) at 5–10 crypts/μl and cultured in a modified form of medium as described in22
. Briefly, DMEM/F12 (Gibco) was supplemented by EGF 40 ng/ml (R&D), Noggin 200 ng/ml (Peprotech), R-spondin 500 ng/ml (R&D or Sino Biological), N-Acetyl-L-cysteine 1 μM (Sigma-Aldrich) and Y-27632 dihydrochloride monohydrate 20 ng/ml (Sigma-Aldrich). cADPR (Sigma), when indicated, was added to culture at 50 μM. 30–50 μl drops of matrigel with crypts were plated onto a flat bottom 48-well plate (Corning 3548) and allowed to solidify for 20 to 30 minutes in a 37° C incubator. 350 μl of crypt culture medium was then overlaid onto the matrigel, changed every other day, and maintained at 37°C in fully humidified chambers containing 6% CO2. Clonogenicity (colony-forming efficiency) was calculated by plating 50 to 400 crypts and assessing organoid formation 3 to 7 days after initiation of cultures.
Culture of isolated cells in supplemented crypt culture medium
Isolated ISCs or Paneth cells were centrifuged for 5 minutes at 250 g and then resuspended in the appropriate volume of crypt culture medium (500–1000cells/μl) supplemented with 1× N2 (Invitrogen), 1× B27 (Invitrogen), 100 ng/ml Wnt-3A (R&D), and with additional 500 ng/ml R-Spondin to yield 1 μg/ml final concentration. ISCs were then seeded into matrigel (BD Bioscience 356231 growth factor reduced) containing 1 μM Jagged (Ana-Spec) up to 5,000–10,000 cells/30–50 μl. 30 μl drops of 65% matrigel were plated onto a flat bottom 48-well plate (Corning 3548) and then Paneth cells were added at the same cell count to the top of the matrigel drop. Alternatively, ISCs and Paneth cells were mixed after sorting in a 1:1 ratio, centrifuged, and then seeded into matrigel. The matrigel drops with ISCs and Paneth cells were allowed to solidify for 20 to 30 minutes in a 37° C incubator. 350 μl of crypt culture medium was then overlayed onto the drops of matrigel and maintained at 37°C in fully humidified chambers containing 6% CO2. The crypt media was changed every second day. Organoid bodies were quantitated on days 3, 7, and 9 days of culture, unless otherwise specified. In subcloning experiments, either individual or cultures of organoids were manually disrupted as indicated in the text on day 7–9 by rigorous tituration and replated into fresh matrigel; these secondary organoid bodies were quantitated on day 18 after initiation of the primary cultures. When indicated, crypts were transfected with 100nM siRNAs targeting the Bst-1 (Thermo Scientific, J-044021-11 and J-044021-12) using the X-Tremegene siRNA transfection reagent, by incubating the crypts at +37°C for 30 min with transfection mixture in crypt medium before mounting to matrigel.
Microarray analysis and validation
Approximately 100,000 Paneth cells were harvested directly to the RLT buffer of the RNeasy plus extraction kit (Qiagen) by the flowcytometry isolation protocol. Total RNA extracts were subjected to microarray analysis by standard protocols of the Whitehead Institute Genome Technology Core (http://jura.wi.mit.edu/genomecorewiki/index.php/Main_Page
) using GeneChip Mouse Gene 1.0 ST arrays (Affymetrix). Expression analysis was conducted with the help of Whitehead Institute Bioinformatics and Research Computing. Briefly, CEL-files were preprocessed with RMA using Bioconductor and the package oligo, and differential expression was assayed by moderated t-test, as implemented by limma. Expression changes were validated by qRT-PCR using oligos:
- Bst-1 ACCCCATTCCTAGGGACAAG, GCCTCCAATCTGTCTTCCAG,
- Wnt3a GGGAGAAATGCCACTGTGTT, TCTCCGCCCTCAAGTAAGAA,
- Myc TCTCCACTCACCAGCACAAC, TCGTCTGCTTGAATGGACAG,
- Gapdh TGTTCCTACCCCCAATGTGT, TGTGAGGGAGATGCTCAGTG
Immediately after removal from the animal, 1.0 mm sections of mouse intestine were placed into Karnovsky's KII Solution (2.5% glutaraldehyde, 2.0% paraformaldehyde, 0.025% calcium chloride, in a 0.1 M sodium cacodylate buffer, pH 7.4), fixed overnight at 4° C, and stored in cold buffer. Subsequently, they were post-fixed in 2.0% osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in graded ethanol solutions, infiltrated with propylene oxide/Epon mixtures, flat embedded in pure Epon, and polymerized over night at 60°C. One micron sections were cut, stained with toluidine blue, and examined by light microscopy. Representative areas were chosen for electron microscopic study and the Epon blocks were trimmed accordingly. Thin sections were cut with an LKB 8801 ultramicrotome and diamond knife, stained with Sato's lead, and examined in a FEI Morgagni transmission electron microscope. Images were captured with an AMT (Advanced Microscopy Techiques) 2K digital CCD camera.