Download APPENDIX Table of content Appendix Supplementary Figures and

APPENDIX
Table of content
Appendix Supplementary Figures and Figure legends
Figure S1
Page 2-3
Figure S2
Page 3-4
Figure S3
Page 5
Figure S4
Page 6
Figure S5
Page 7
Appendix Supplementary Methods
Isolation of embryonic intestinal epithelial cells using flow cytometry
Page 8
Isolation of adult ISCs using flow cytometry
Page 8
RNA-sequencing data analyses
Page 9
Histological techniques
Page 10
RNA in situ hybridization
Page 11
Cell culture, luciferase and western blot assays
Page 11
Supplementary References
Page 12
1
Appendix Figure S1 - Defining molecular signatures of the embryonic intestinal epithelium
and adult ISCs.
(A-B) FACS strategy used to isolate EpCAM+ cells from the embryonic small intestine at E11.5
and Lgr5-EGFPhigh cells from the adult intestinal crypts. (A) Cells from the dissected embryonic
small intestines were dissociated by enzymatic digestion and stained with EpCAM antibody.
These cells were selected based on size (P1 and P2), DAPI dye exclusion (P3, living cells) and
2
EpCAM+ labelling (P4). (B) Cells from the adult intestinal crypts of Lgr5EGFP-Cre-ERT mice were
dissociated by enzymatic digestion. Single living Lgr5-EGFPhigh cells were selected based on size
(P1 and P2), DAPI dye exclusion (P3, living cells), and the level of EGFP expression (P4). (C-H)
UCSC Genome Browser images of RNA-seq data, with transcript profiles of polyA+ mRNA from
embryonic small intestinal epithelium (orange) and the adult ISCs (green). Bambi, Fgfr2 and
Igfbp2 are expressed in the embryonic small intestine (C-E), whereas Cd44, Smoc2 and Olfm4
were detected in the adult ISCs only (F-H). The y-axis indicates the coverage normalized by
library size (reads per million).
Appendix Figure S2 - Strategies for labelling and quantification of various cell populations
within the embryonic small intestine.
(A-B) FACS strategy used to analyse Lgr5-EGFP+ cells from the embryonic small intestine at
various developmental stages. Cells from the dissected small intestines either of wild type (A) or
Lgr5EGFP-Cre-ERT (B) embryos were dissociated by enzymatic digestion and stained with EpCAM
antibody. The cells were selected based on size (P1 and P2), DAPI dye exclusion (P3, living
cells), EpCAM+ labelling (P4) and expression of EGFP (P5). (C) FACS strategy used to analyse
Lgr5-EGFP+:tdTomato+ positive cells from the small intestine of Lgr5EGFP-Cre-ERT/Rosa26tdTomato
embryos after tamoxifen treatment. The cells were selected based on size (P1 and P2), DAPI dye
exclusion (P3, living cells), EpCAM+ labelling (P4) and expression of either EGFP (P5) or
tdTomato (P6). (D, F) FACS strategy used to analyse tdTomato+ positive cells from the small
intestine
of
Rosa26Cre-ERT/Rosa26tdTomato
embryos
after
tamoxifen
treatment.
(E,
G)
Representative section of small intestine from Rosa26Cre-ERT/Rosa26tdTomato mouse embryos
showing ubiquitous labelling of Rosa-Cre-ERT+ cells one day after single treatment with TAM at
E13.5 (n=3 mice analysed). Scale bar: 30 µm (E-G).
3
4
Appendix Figure S3 - Molecular signature of embryonic Lgr5+ progenitors.
(A-B) FACS strategy used to isolate Lgr5+EpCAM+ cells from the embryonic small intestine at
E13.5. Cells from the dissected small intestines of either wild type (A) or Lgr5EGFP-Cre-ERT (B)
embryos (n³10) were dissociated by enzymatic digestion and stained with EpCAM antibody. The
cells were selected based on size (P1 and P2), DAPI dye exclusion (P3, living cells), EpCAM+
labelling (P4) and expression of EGFP (P5). (C-F) Sections of small intestines from embryos at
E13.5 (C, E) and adult mice at P60 (D, F) stained with anti-Cd44 (C-D) or anti-Lrig1 (E-F)
antibodies. (G-H) LacZ staining of small intestines from Prom1LacZ-Cre-ERT embryos at E13.5 (G)
and adult mice at P60 showing the activity of Prom1 promoter. Scale bar: 100µm (C, E), 15µm
(D, F, H) and 50 µm (G).
5
Appendix Figure S4 - Id2 functions in the intestinal epithelium.
(A) qRT-PCR analysis for selected genes in Id2KO (blue) and wild type (red) mesenchymal cells
at E11.5. Tbp expression was used as normalizing control. Error bars are ± SD, (n=3).
***P<0.001 by Student´s t-test.
6
Appendix Figure S5 - Ectopic Wnt ligands activate Wnt/-catenin signalling in Id2 mutant
intestinal epithelium.
(A-F) Representative FACS plots showing strategy to quantify Lgr5-EGFP+EpCAM+ cells
(green) in control (A, D), Lgr5EGFP-CRE-ERT (B, E) and Id2KO:Lgr5EGFP-CRE-ERT embryos (C, F) at
E15.5 after administration of vehicle (A-C) or Wnt-C59 inhibitor (D-F) (n=3). (G-I)
Representative FACS plots showing strategy to quantify Lgr5-EGFP+EpCAM+ cells (green) in
control (G) and Id2KO:Lgr5EGFP-CRE-ERT embryos at E11.5 after administration of vehicle (H) or
Wnt-C59 inhibitor (I) (n=3).
7
APPENDIX SUPPLEMENTARY METHODS
Isolation of embryonic intestinal epithelial cells using flow cytometry
Small intestines were dissected from mouse embryos at indicated stages, cut in pieces of 2 mm
and incubated for 5-10 min with 0.15mg/ml collagenase (Sigma) in PBS at 37°C with shaking at
800 rpm. Single cell suspensions were collected by centrifugation at 200g for 3 min, washed
twice and resuspended in PBS supplemented with 2% foetal calf serum. Cells were stained with
PerCP-eFluor® 710–conjugated anti-EpCAM 1:400 (17-5791-82, eBioscience), PE-conjugated
anti-CD45 at 1:400 (553081, BD Biosciences) and anti-CD31 at 1:400 (561073, BD Biosciences)
antibody for 15 min at room temperature. Living cells were gated by DAPI dye exclusion.
Embryonic intestinal epithelial cells were isolated as EpCAM+CD31 CD45 DAPI . Embryonic
Lgr5+ cells were isolated as EGFP+EpCAM+CD31 CD45 DAPI . Fluorescence-activated cell
sorting analysis was performed using BD FACS Aria III SORP cell sorter (85 M nozzle) and
FlowJo software.
Isolation of adult ISCs using flow cytometry
Small intestines were dissected from 2 months old Lgr5EGFP-Cre-ERT males, cut in pieces of 2 mm2,
placed into 50 ml conical tubes and washed 3 times for 10 min with 40 ml of PBS/ 5 mM EDTA
on a rocking plate at room temperature. Villi were mechanically removed by gentle shaking 3
times and trituration using a 10 ml pipette. Crypts were separated from the intestine by vigorous
shaking 2 times passing through a 70 M filter (BD Falcon). Crypt material was collected by
centrifugation at 200g for 3 min, washed twice with PBS and incubated for 5-10 min with 0.15
mg/ml collagenase (Sigma) and 0.1 mg/ml DNase I (Qiagen) at 37°C on a rocking plate. Single
cell suspensions were washed twice and re-suspended in PBS supplemented with 2% foetal calf
serum. Cells were stained with PE-conjugated anti-CD45 at 1:400 (BD Biosciences) and anti8
CD31 at 1:400 (BD Biosciences) antibody for 30 min at room temperature. Living cells were
gated by DAPI dye exclusion. ISCs were isolated as Lgr5-EGFPhighCD31 CD45 DAPI .
Fluorescence-activated cell sorting analysis was performed using BD FACS Aria III SORP cell
sorter (85 M nozzle) and FlowJo software.
RNA-sequencing data analyses
Raw
reads
were
pre-processed
and
the
quality
was
assessed
with
FastQC
(http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). Reads were mapped to the iGenomes
(http://support.illumina.com/sequencing/
sequencing_software/igenome.html)
UCSC
mouse
genome reference version mm9 using STAR version 2.4.0h (Dobin et al, 2013) as the aligner,
allowing up to two mismatches and keeping only the primary reads. Reads mapping to more than
10 loci were discarded, and the minimum intron size was set to 10 nucleotides. The junctions’
database for the STAR reference was built with the gene model from the iGenomes, downloaded
from UCSC. Reads falling on features were counted with the HTSeq-count (Anders et al, 2015)
software, using the same gene model used before to guide the aligner, and handling overlapping
reads in the default union mode. For the differential expression analysis, we used R
(http://www.R-project.org/) and DESeq2 (Love et al, 2014) to normalize, transform, and model
the data. The counts were fitted to a Negative Binomial generalized linear model (GLM) and the
Wald significance test was used to determine the differentially expressed genes between various
epithelial cell populations. Finally, RPKM values were calculated per gene using the library sizenormalized FPM (robust counts per million mapped fragments) values from DESeq2 and
multiplying them by 1000 divided by the gene length. Gene Set Enrichment Analysis was
performed with the web service DAVID (Huang et al, 2007).
9
Histological techniques
Embryonic or adult small intestines were dissected, fixed for 1 hour in 1% formaldehyde in PBS
at 4°C, incubated overnight in 30% sucrose, embedded in OCT and kept at -80°C.
Immunohistochemical analyses were performed on 10 m cryosections using 1:1000 anti-EpCAM
antibody (17-5791-82, eBiosciences), 1:50 anti-Trop2 (C-12, Santa Cruz), 1:100 anti-Lrig1
(AF3688, R&D), 1:500 CD44v6 (BMS145, eBioscience) followed by 1:300 Alexa566conjugated secondary antibodies (Invitrogen), and anti-Chromogranin A 1:1000 (20085,
ImmunoStar Inc.) followed by Vectastain Elite ABC kit (Vector Laboratories) according to the
manufacturer’s instructions. Lgr5-EGFP was detected using anti-GFP antibody 1:2000 (A-11122,
Thermofisher Scientific) followed by Alexa488-conjugated goat anti-rabbit antibody 1:300
(Invitrogen). Counterstaining of nuclei was performed with DAPI 1:1000 (Sigma). EdU was
detected using the Click-iT® EdU Flow Cytometry Assay Kit according to the manufacturer’s
instructions (Thermofisher). Periodic Acid Schiff staining was performed according to the
manufacturer’s instructions (Sigma).
To detect LacZ+ cells tissue sections were fixed with 1% formaldehyde in PBS for 20
min, washed 3 times with PBS/ 0.01% NP-40/ 0.01% Na-deoxycholate and incubated overnight
in staining solution (5mM K3[Fe(CN)6], 5mM K4[Fe(CN)6], 2mM MgCl2, 0.01% NP-40, 0.01%
Na-deoxycholate and 1mg/ml X-gal in PBS). For whole mount LacZ staining, small intestines
were isolated and fixed with 0.2% glutaraldehyde in PBS for 20 min, washed 3 times with PBS/
0.01% NP-40/ 0.01% Na-deoxycholate and incubated 4 hours in the staining solution. Images
were acquired with confocal Leica TSC SP5, Leica DM2500, Leica AF7000 and Leica M205FA
microscopes.
10
RNA in situ hybridization
Embryonic small intestines were dissected, fixed overnight in 4% formaldehyde in PBS at 4°C,
dehydrated and embedded in paraffin. 10 m sections were rehydrated, treated 15 min with 6%
H2O2, 10 min with 20
g/ml proteinase K, post-fixed with 4% formaldehyde for 15 min,
demethylated with acetic anhydride, equilibrated with 2x SSC and hybridized overnight at 63°C
with digoxygenin labelled RNA probes for Id2, Lgr5, Axin2, Sfrp5, and Shh. Sections were
washed and incubated overnight with sheep anti-digoxygenin antibody 1:3000 (Roche). The
signal was revealed using NBT/BCIP (Roche). Images were acquired with Leica DM2500
microscope.
Cell culture, luciferase and western blot assays
HEK293T cells were grown at 370C in DMEM (Gibco) media supplemented with 10% FBS
(Gibco), 2 mM Glutamax (Gibco), non-essential amino acids (Gibco), 100 U/ml penicillin and
100 g/ml streptomycin (Gibco). Cells were transfected using Turbofect (Thermofisher
Scientific) according to the manufacture instructions. Analyses were performed 48 hours posttransfection. TOP (#12456) and FOP (#12457) plasmids were obtained from Addgene. The
luciferase reporter activity was measured using Dual-Luciferase Reporter Assay System
(Promega). Samples were analysed by 9% SDS-PAGE and transferred to PVDE membrane.
Membranes were immunoblotted using FLAG M2 antibodies 1:1000 (F3165, Sigma).
Horseradish peroxidase-conjugated anti-mouse IgG (NEB) were used at a dilution 1:3000. HRPchemiluminescence was developed using Western Lightning Chemiluminescence Reagent Plus
(PerkinElmer).
11
SUPPLEMENTARY REFERENCES
1.
Anders S, Pyl PT, Huber W (2015) HTSeq--a Python framework to work with highthroughput sequencing data. Bioinformatics 31: 166-169
2.
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M,
Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29: 15-21
3.
Huang DW, Sherman BT, Tan Q, Collins JR, Alvord WG, Roayaei J, Stephens R, Baseler
MW, Lane HC, Lempicki RA (2007) The DAVID Gene Functional Classification Tool: a
novel biological module-centric algorithm to functionally analyze large gene lists. Genome
Biol 8: R183
4.
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion
for RNA-seq data with DESeq2. Genome Biol 15: 550
12