supplemental material small intestinal stem cell identity...

Supplemental Material

Small Intestinal Stem Cell Identity Is Maintained with Functional Paneth Cells

in Heterotopically Grafted Epithelium onto Colon

Masayoshi Fukuda, Tomohiro Mizutani, Wakana Mochizuki, Taichi Matsumoto, Kengo Nozaki, Yuriko Sakamaki, Shizuko Ichinose,

Yukinori Okada, Toshihiro Tanaka, Mamoru Watanabe, Tetsuya Nakamura

Supplemental Materials and Methods Supplemental Figures and Figure Legends: Supplemental Figure S1 Colonic epithelial injury model used in the present study Supplemental Figure S2 Freshly isolated SI epithelia also maintain SI phenotype when grafted Supplemental Figure S3 Subepithelium of the graft contains multiple cellular components of recipient origin Supplemental Figure S4 Multi-differentiation and self-renewal capabilities of grafted cells at 4 months post-transplantation Supplemental Figure S5 Laser capture microdissection and following microarray analysis Supplemental Table S1 List of genes differentially expressed between the graft and normal colonic epithelium

Supplemental Materials and Methods

Stereomicroscopy, Histology, and Immunohistochemistry

Whole distal colons of recipients and their fluorescence were imaged using a fluorescence stereomicroscope

system MVX10 (Olympus). For histology and immunohistochemistry, recipient colons were fixed overnight at 4˚C

in 4% paraformaldehyde, sequentially dehydrated in 10, 15 and 20% sucrose in PBS, and embedded in OCT

compound (Tissue Tek). Frozen sections of 6-mm thickness were cut, mounted on slide glasses, and examined

for their fluorescent signals in order to determine whether the section contains EGFP+ grafts. Sections containing

EGFP+ grafts were subjected to H&E staining or immunohistochemistry. The followings were used as antibodies

specific for each protein: Cdh (Cell Signaling); CA2 (Santa Cruz); CDX2 (Bio Genex); Ki67 (Dako Cytomation);

MUC2 (Santa Cruz); ChgA (ImmunoStar); sucrose isomaltase (Santa Cruz); Lysozyme (DAKO); CD3 (BD); F4/80

(Abcam); and CD31 (Abcam). In all immunofluorescent experiments, nuclei were counterstained with DAPI.

Sections obtained from some tissue blocks showed obvious but weak endogenous EGFP signals presumably

because of the variation in the rate and quality of fixation. In those cases, we performed immunofluorescence

staining of adjacent sections with anti GFP antibody (Invitrogen) and presented them to show the EGFP

fluorescence as a reference. Fluorescent images of sections were acquired using a DeltaVision system (Applied

Precision) where a fluorescent microscope IX-71 (Olympus) with objectives UplansApo 10x 0.4NA or UplansApo

20x 0.75NA (Olympus) is incorporated. If necessary, image processing was carried out using Adobe Photoshop

Elements 7.0 software. For quantification of cellular components, sections of proximal, middle, distal SI, colon,

and EGFP+ grafts were subjected to immunohistochemistry for ChgA, MUC2, or Lysozyme. Thirty crypt-villus

units or crypt units (colon), originating from 3 independent recipients or normal controls, were analyzed. The

numbers of ChgA+, MUC2+, or Lysozyme+ cells were counted and presented as mean cell counts per crypt-villus

or crypt unit. Statistical significance was determined by Student’s t test (p < 0.05).

Laser Capture Microdissection

Recipient mice at 4 weeks post-transplantation and C57BL normal control mice were sacrificed. The

graft-containing recipients’ tissues and control colon tissues were quickly washed in PBS, embedded in OCT

compound (Tissue Tek), rapidly frozen, and stored. For recipients’ colon experiments, we searched and located

the plane of specimens so that the section would contain EGFP+ grafts. From there onwards, serial cryosections

(10-mm thickness) of more than 30 slices were made. Fluorescent images of a part of these slices (1 every 7

sections) were acquired, and they were used as references for dissecting the EGFP+ epithelia from sections

located in between. Likewise, we also made ~ 30 sections from the distal colon of control mice. Sections were

stained with cresyl violet solution and Arcturus HistGene Staining Solution (Applied Biosystems). Grafted

epithelia in recipients or control colonic epithelia were microdissected using Micro Laser System MBIII

(PALM/ZEISS). Dissected epithelia obtained from serial sections (~ 30 sections) of each specimen were

combined and then total RNA extraction was performed by using RNeasy Plus Micro Kit (QUIAGEN). After

calculating RNA Integrity Number (RIN), we subjected one graft sample and one control colon sample, which

showed the highest RIN value, for the following gene expression analysis.

Microarray and Data Analysis

cDNA synthesis, amplification and labeling were performed with Ovation Pico WTA System V2 and Encore Biotin

Module (Nugen). Microarray hybridization was carried out onto GeneChip Mouse Gene 1.0 ST Array (Affymetrix).

The arrays were scanned with GeneChip Scanner 3000 7G (Affymetrix). Data files were imported to GeneSpring

GX 12.5 software (Agilent), and its Robust Multichip Average algorithm 16 (RMA16) was used for data

normalization. The bottom 20% of genes with the lowest expression levels was excluded from subsequent

analysis. Multiple probesets on Affymetrix microarrays are associated with transcript clusters. Transcript clusters

that showed > 3-fold differences in expression values between two samples are presented. For a gene or a group

of genes to which values are given by multiple transcript clusters, averages of the values for those clusters were

used for assessing fold enrichment. Detailed information on the probeset and transcript cluster grouping is

available on the NetAffyx site (http://www.affymetrix.com/estore/index.jsp) provided by the manufacturer.

Fukuda et al., Supplemental Figure S1

A

B

Supplemental Fig. S1 Colonic epithelial injury model used in the present study.(A) Devices used to generate distal colonic epithelial injury. Thin catheter equipped with two small rubber balloons used for topical EDTA treatment (left). After the EDTA treatment, balloons were deflated and the catheter was removed out of the colon. Epithelial abrasion was performed by using an electric brush (right), giving its rotational movement to scratch the colonic luminal surface. (B) Time course change of the tissue damage during recovery. Stereoscopic images (left) at Day 2, 14, 28 after injury are presented with their longitudinal sections (right). H&E-stained sections are shown with anal side on the left and oral side on the right. High-power views of areas in dotted rectangles are shown at the bottom. Scale bars show 1 mm.

Day 2

Day 14

Day 28

Day 2

Day 14

Day 28


Supplemental Fig. S2Freshly isolated SI epithelia also maintain SI phenotype when grafted. SI epithelia were isolated from EGFP transgenic mice and transplanted immediately into recipients without culturing. Recipient colons at 2 weeks post-transplantation were analyzed. (A) immunostaining for Lysozyme (A) and endogenous EGFP fluorescence (A´) of the same section. (B) Section double stained with CDX2 (B) and CA2 (B´) is presented with the endogenous EGFP fluorescence of the same section (B´´). Note that the antigen retrieval procedure before immunostaining completely clears endogenous EGFP signals.Images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm.

EGFP

A´�A�

Lysozyme

B´�B� B´´�

CDX2 EGFPCA2

Supplemental Fig. S3Subepithelium of the graft contains multiple cellular components of recipient origin. Immunohistochemical analysis of recipient colons at 4 weeks post-transplantation. Tissue sections were immunostained with anti-CD3 (A), anti-F4/80 (B), or anti-CD31 (C) antibody to detect T cells, monocyte/macrophage lineage of cells, and blood vessel endothelial cells, respectively. EGFP fluorescence of the same sections (A´, B´, and C´) is also shown. Arrowheads point to positively-stained cells residing within the subepithelia entirely surrounded by EGFP+ epithelia. Images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm.


EGFPCD31

EGFPCD3

EGFPF4/80

C´�

B´�

A´�

C�

B�

A�


Supplemental Fig. S4 Multi-differentiation and self-renewal capabilities of grafted cells at 4 months post-transplantation. (A) H&E staining of a section obtained from a recipient colon at 4 months (18 weeks) post-transplantation. (B,C) EGFP fluorescence (B) of the section adjacent to the one shown in (A) and its immunostaining for Lysozyme (C). High-power views of areas in dotted boxes in (A) and (C) are shown as (A´) and (C´), respectively. (D) Section double stained with CA2 (D) and sucrase isomaltase (SIase; D´) is presented with the endogenous EGFP fluorescence of the same section (D´´). Note that the antigen retrieval procedure before immunostaining completely clears endogenous EGFP signals. (E-H) Immunofluorescence for CDX2 (E), Ki67 (F), MUC2 (G), and ChgA (H). Arrowheads point to ChgA+ cells. Images of endogenous EGFP fluorescence of the same sections (E´, F´, G´) or an adjacent section (H´) are shown. Fluorescent images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm.

Ki67

CA2EGFP

Lysozyme

C´�

C�

Lysozyme

EGFP

SIase

F´�

F�

G´�

G�

EGFP EGFP

MUC2

D´´�

H´�

H�

EGFP

ChgA

E´�

E� CDX2

EGFP

A�

B�

A´�

D´�

D�

RNA Extraction

cDNA Amplification

Microarray Analysis

Post Dissection

Pre Dissection

Mesenchyme elimination

EGFP

Pre Dissection

Post Dissection

Graft Control Colon

Supplemental Fig. S5 Laser capture microdissection and following microarray analysis. The graft-containing tissues and control colon tissues were obtained from recipient mice (4 weeks post-transplantation) and normal syngeneic mice, respectively. To obtain donor-derived epithelia from recipients’ colon, serial sections were prepared per sample so that all sections contain EGFP+ epithelia. Every 6th section was used to detect EGFP+ signals as references for dissecting the EGFP+ epithelia from other sections located in between. Microdissected epithelia from multiple sections of each sample were combined and then total RNA was extracted. RNA was converted to cDNA, amplified, labeled, and used for GeneChip hybridization. Scale bars show 100 μm.


No Affimetrix Transcripts Cluster ID Gene Symbol Gene Description Fold Change Graft/Colon1 10550131 Pla2g4c phospholipase A2, group IVC (cytosolic, calcium-independent) 129.04922 10419575 Ang4 angiogenin, ribonuclease A family, member 4 111.4434

Defa24 defensin, alpha, 24Defa6 defensin, alpha, 6Defa23 defensin, alpha, 23

Defa-rs7 defensin, alpha, related sequence 7Defa17 defensin, alpha, 17Defa3 defensin, alpha, 3

Gm15284 predicted gene 15284Gm15284 predicted gene 15284

Defa17 defensin, alpha, 17Defa3 defensin, alpha, 3Defa24 defensin, alpha, 24Defa6 defensin, alpha, 6Defa23 defensin, alpha, 23


Gm15284 predicted gene 15284Defa23 defensin, alpha, 23


7 10501026 Chi3l4 chitinase 3-like 4 60.58008 10538965 Fabp1 fatty acid binding protein 1, liver 50.76149 10356367 Alpi alkaline phosphatase, intestinal 48.5584

1058501010593174

11 10436087 Retnlb resistin like beta 47.610312 10585005 Apoa1 apolipoprotein A-I 46.774413 10498659 Sis sucrase isomaltase (alpha-glucosidase) 43.769714 10558590 Prap1 proline-rich acidic protein 1 42.009515 10416689 Olfm4 olfactomedin 4 34.035516 10502613 AI747448 expressed sequence AI747448 31.730617 10464328 Pnliprp2 pancreatic lipase-related protein 2 29.7698

Itln1 intelectin 1 (galactofuranose binding) Itlnb intelectin b

19 10347117 Cps1 carbamoyl-phosphate synthetase 1 25.037220 10430425 Lgals2 lectin, galactose-binding, soluble 2 22.8981

Defa-ps1 defensin, alpha, pseudogene 1Defa25 defensin, alpha, 25Defa17 defensin, alpha, 17Defa3 defensin, alpha, 3Defa23 defensin, alpha, 23

Defa-rs7 defensin, alpha, related sequence 7Gm15299 predicted pseudogene 15299

Defa2 defensin, alpha, 2Gm15292 predicted gene 15292

Defa26 defensin, alpha, 2622 10372652 Lyz1 lysozyme 1 19.8162

Defa25 defensin, alpha, 25Gm15299 predicted pseudogene 15299Gm6696 predicted gene 6696Defa-ps1 defensin, alpha, pseudogene 1Defa23 defensin, alpha, 23

24 10583133 Mmp7 matrix metallopeptidase 7 18.627825 10402708 Ckb creatine kinase, brain 18.123226 10531407 Cxcl9 chemokine (C-X-C motif) ligand 9 17.041327 10384102 Npc1l1 NPC1-like 1 16.160028 10502375 Mttp microsomal triglyceride transfer protein 14.5011

1059548010405753

30 10456005 Cd74 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) 12.803731 10368317 Enpp3 ectonucleotide pyrophosphatase/phosphodiesterase 3 12.522632 10549154 Gm766 predicted gene 766 11.736033 10543411 Slc13a1 solute carrier family 13 (sodium/sulfate symporters), member 1 11.654234 10438567 2310042E22Rik RIKEN cDNA 2310042E22 gene 11.543135 10444291 H2-Ab1 histocompatibility 2, class II antigen A, beta 1 11.462336 10489391 Ada adenosine deaminase 11.360837 10475448 Duoxa2 dual oxidase maturation factor 2 10.920638 10445112 Ubd ubiquitin D 10.415739 10545208 Gm189 predicted gene 189 10.305240 10369567 Hkdc1 hexokinase domain containing 1 9.7970

Arg2 arginase type II Vti1b vesicle transport through interaction with t-SNAREs 1B homolog

Defa25 defensin, alpha, 25

41 10396831 9.7368

23 10570700 19.0097

29 Me1 malic enzyme 1, NADP(+)-dependent, cytosolic 13.5514

18 10360149 29.4274

21 10570683 20.8023

10570668 71.0562

6 1057069010570720 60.7345

10 Apoa4 apolipoprotein A-IV 48.3594

Supplemental Table S1. List of genes differentially expressed between the graft and normal colonicepithelium.Listed in this table are the transcript clusters (See Experimental Procedures) that showed > 3-folddifferences in expression values between two samples. For a gene or a group of genes to which values aregiven by multiple transcript clusters, the average of values for those clusters was used for assessing foldenrichment and presented.

List of genes enriched in Graft

3 10570735 102.2967

4 1057065610570676 73.6512

5

Gm6696 predicted gene 6696Defa-ps1 defensin, alpha, pseudogene 1Gm15299 predicted pseudogene 15299

Defa23 defensin, alpha, 2343 10386197 2210407C18Rik RIKEN cDNA 2210407C18 gene 8.824444 10552193 Slc7a9 solute carrier family 7 (cationic amino acid transporter, y+ system), member 9 8.7444

Defa20 defensin, alpha, 20Gm15308 predicted gene 15308

Defa3 defensin, alpha, 346 10407337 Hcn1 hyperpolarization-activated, cyclic nucleotide-gated K+ 1 8.697947 10486956 Duox2 dual oxidase 2 8.602448 10372557 Gm239 predicted gene 239 8.4779

Tgtp1 T-cell specific GTPase 1Tgtp2 T-cell specific GTPase 2

Gm12185 predicted gene 12185Defa26 defensin, alpha, 26Defa23 defensin, alpha, 23

Defa-rs7 defensin, alpha, related sequence 751 10384044 Myl7 myosin, light polypeptide 7, regulatory 7.967552 10539200 Reg1 regenerating islet-derived 1 7.957353 10405587 Tgfbi transforming growth factor, beta induced 7.800754 10425037 Apol10a apolipoprotein L 10a 7.663155 10588043 Rbp2 retinol binding protein 2, cellular 7.431556 10539179 Reg3b regenerating islet-derived 3 beta 6.938557 10419563 Rnase1 ribonuclease, RNase A family, 1 (pancreatic) 6.873858 10380581 Gip gastric inhibitory polypeptide 6.754359 10394394 Apob apolipoprotein B 6.555660 10527638 Alox5ap arachidonate 5-lipoxygenase activating protein 6.2527

1048972110363561104273891056559810424377104222471056853410528205106035491050052710410927105810091047874610589974105281651054112910543029103454361039527510346222104576671048435510607429104831611049732710586863104684871046912710374352103741831035676210540531105274251060380310357298104763991052274210363005105744321057443410496167105829831051246310496336103596421044823010416698104853551041669610375324

62 10377841 Tm4sf5 transmembrane 4 superfamily member 5 6.1292Defa5 defensin, alpha, 5

Gm15315 predicted gene 15315Gm14850 predicted gene 14850Gm10104 predicted gene 10104Gm15293 predicted gene 15293

64 10420747 Gata4 GATA binding protein 4 6.091365 10455961 Iigp1 interferon inducible GTPase 1 6.073166 10388902 Lgals9 lectin, galactose binding, soluble 9 6.0658

Gm1943 WD repeat domain 70 pseudogeneWdr70


Gm14850 predicted gene 14850Gm10104 predicted gene 10104Gm15293 predicted gene 15293

Defa4 defensin, alpha, 41040303410403063

68 10570732 5.9227

63 10570693 6.1097

69 LOC100046275 ig heavy chain V-II region SESS-like 5.6599

67 10581575 6.0203

50 10570726 7.9827

61 LOC280487 pol polyprotein 6.1985

45 1057071110570706 8.7175

49 1038551810385533 8.4646

42 10570663 9.1532

70 10603066 Ace2 angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 5.656171 10358399 Rgs13 regulator of G-protein signaling 13 5.6483

Gm10104 predicted gene 10104 Gm14850 predicted gene 14850

Defa5 defensin, alpha, 5Gm15315 predicted gene 15315 Gm15293 predicted gene 15293

Defa4 defensin, alpha, 4Gm14850 predicted gene 14850Gm10104 predicted gene 10104

Defa5 defensin, alpha, 5Gm15315 predicted gene 15315Gm15293 predicted gene 15293

Defa4 defensin, alpha, 474 10363372 Pla2g12b phospholipase A2, group XIIB 5.4279

Chn2 chimerin (chimaerin) 29130019P16Rik RIKEN cDNA 9130019P16 gene

76 10546929 Cidec cell death-inducing DFFA-like effector c 5.398777 10570018 Tnfsf13b tumor necrosis factor (ligand) superfamily, member 13b 5.279478 10546685 Eif4e3 eukaryotic translation initiation factor 4E member 3 5.248479 10543319 Fam3c family with sequence similarity 3, member C 5.1963

Defa22 defensin, alpha, 22Defa21 defensin, alpha, 21

81 10457475 Abhd3 abhydrolase domain containing 3 4.980282 10444298 H2-Eb1 histocompatibility 2, class II antigen E beta 4.963683 10538871 Gm4964 predicted gene 4964 4.8887


Gm15299 predicted pseudogene 15299Defa-ps1 defensin, alpha, pseudogene 1

85 10489759 Sulf2 sulfatase 2 4.8775Defa21 defensin, alpha, 21Defa22 defensin, alpha, 22

87 10542857 Far2 fatty acyl CoA reductase 2 4.857888 10362674 Rnu3a U3A small nuclear RNA 4.847589 10560719 2210010C17Rik RIKEN cDNA 2210010C17 gene 4.829890 10449467 Clps colipase, pancreatic 4.811491 10589166 Slc26a6 solute carrier family 26, member 6 4.774392 10607602 Ppef1 protein phosphatase with EF hand calcium-binding domain 1 4.756593 10408629 1300014I06Rik RIKEN cDNA 1300014I06 gene 4.744894 10424430 Gsdmcl-ps gasdermin C-like, pseudogene 4.7265

1035464910354647

96 10545425 Sh2d6 SH2 domain containing 6 4.6303B4galnt1 beta-1,4-N-acetyl-galactosaminyl transferase 1Slc26a10 solute carrier family 26, member 10

98 10444229 H2-DMa histocompatibility 2, class II, locus DMa 4.568399 10465831 5730408K05Rik RIKEN cDNA 5730408K05 gene 4.5082100 10593473 1810046K07Rik RIKEN cDNA 1810046K07 gene 4.5000101 10429926 Dgat1 diacylglycerol O-acyltransferase 1 4.4767102 10511429 Car8 carbonic anhydrase 8 4.4738

Defa25 defensin, alpha, 25Gm6696 predicted gene 6696Gm15299 predicted pseudogene 15299

104 10569962 Ccl25 chemokine (C-C motif) ligand 25 4.4339105 10348451 Cxcr7 chemokine (C-X-C motif) receptor 7 4.4174106 10422760 Fyb FYN binding protein 4.4159107 10428955 Gsdmc2 gasdermin C2 4.3435108 10495820 Fabp2 fatty acid binding protein 2, intestinal 4.3159109 10355403 Fn1 fibronectin 1 4.3106110 10572271 Tm6sf2 transmembrane 6 superfamily member 2 4.2767111 10408613 Tubb2b tubulin, beta 2B 4.2559112 10419198 Ero1l ERO1-like (S. cerevisiae) 4.2502113 10527565 Pdx1 pancreatic and duodenal homeobox 1 4.2063114 10475487 Slc28a2 solute carrier family 28 (sodium-coupled nucleoside transporter), member 2 4.1907115 10498710 Bche butyrylcholinesterase 4.1873116 10383930 Gm10278 predicted gene 10278 4.1861117 10446656 Lpin2 lipin 2 4.1630

Abpd androgen binding protein deltaGm9138 predicted gene 9138

119 10450154 H2-Aa histocompatibility 2, class II antigen A, alpha 4.0963120 10371054 Creb3l3 cAMP responsive element binding protein 3-like 3 4.0921

Defa-rs1 defensin, alpha, related sequence 1Gm14851 predicted gene 14851Gm7849 predicted gene 7849Gm7861 predicted gene 7861

AY761184 cDNA sequence AY761184122 10493831 S100a8 S100 calcium binding protein A8 (calgranulin A) 4.0382

Irgm2 immunity-related GTPase family M member 2Igtp interferon gamma induced GTPase

124 10492174 Tm4sf4 transmembrane 4 superfamily member 4 4.0029125 10516064 Mfsd2a major facilitator superfamily domain containing 2A 3.9983126 10495993 Elovl6 ELOVL family member 6, elongation of long chain fatty acids (yeast) 3.9754

Slc3a1 solute carrier family 3, member 1Prepl prolyl endopeptidase-like

128 10452770 Capn13 calpain 13 3.9264129 10346668 Fam117b family with sequence similarity 117, member B 3.9235130 10578857 Anxa10 annexin A10 3.8842131 10542355 Emp1 epithelial membrane protein 1 3.7811132 10544114 Hipk2 homeodomain interacting protein kinase 2 3.7730133 10430372 Rac2 RAS-related C3 botulinum substrate 2 3.7719134 10461439 Fads1 fatty acid desaturase 1 3.7640135 10409278 Nfil3 nuclear factor, interleukin 3, regulated 3.7625136 10467470 Aldh18a1 aldehyde dehydrogenase 18 family, member A1 3.7439

Gm7849 predicted gene 7849Gm7861 predicted gene 7861Gm14851 predicted gene 14851137 10577434 3.7197

123 10376326 4.0222

127 10447264 3.9326

118 10562291 4.1343

121 10577426 4.0639

97 10366848 4.5800

103 10570723 4.4495

86 10570651 4.8610

95 Pgap1 post-GPI attachment to proteins 1 4.6861

80 10570671 5.1204

84 10570703 4.8801

73 10570717 5.4712

75 10538356 5.4086

72 10570660 5.5796

Defa-rs1 defensin, alpha, related sequence 1AY761184 cDNA sequence AY761184

138 10498978 Lrat lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) 3.6999139 10357488 Cd55 CD55 antigen 3.6812140 10547153 Alox5 arachidonate 5-lipoxygenase 3.6810141 10450800 Trim15 tripartite motif-containing 15 3.6748142 10546962 Ghrl ghrelin 3.6727143 10506433 Dab1 disabled homolog 1 (Drosophila) 3.6018144 10517609 Cda cytidine deaminase 3.5674145 10420988 Dpysl2 dihydropyrimidinase-like 2 3.5322146 10585186 1600029D21Rik RIKEN cDNA 1600029D21 gene 3.5317

1035146310351455

148 10475653 Slc27a2 solute carrier family 27 (fatty acid transporter), member 2 3.5193H2-T3 histocompatibility 2, T region locus 3

H2-T3-like MHC class I antigenH2-D1 histocompatibility 2, D region locus 1H2-T18 histocompatibility 2, T region locus 18

150 10466008 Dak dihydroxyacetone kinase 2 homolog (yeast) 3.4970151 10485405 Cd44 CD44 antigen 3.4922152 10472097 Fmnl2 formin-like 2 3.4921153 10477250 Hck hemopoietic cell kinase 3.4641154 10525016 Tbx3 T-box 3 3.4567155 10444312 Btnl2 butyrophilin-like 2 3.4509156 10440513 Cyyr1 cysteine and tyrosine-rich protein 1 3.4321157 10496023 Casp6 caspase 6 3.4140158 10512904 Aldob aldolase B, fructose-bisphosphate 3.3880159 10393573 Lgals3bp lectin, galactoside-binding, soluble, 3 binding protein 3.3847160 10406176 Slc9a3 solute carrier family 9 (sodium/hydrogen exchanger), member 3 3.3712161 10496638 Odf2l outer dense fiber of sperm tails 2-like 3.3320162 10489204 Tgm2 transglutaminase 2, C polypeptide 3.3276163 10466530 Pcsk5 proprotein convertase subtilisin/kexin type 5 3.3231164 10366774 Avil advillin 3.3180165 10485597 Depdc7 DEP domain containing 7 3.3066

Gm14851 predicted gene 14851Defa-rs1 defensin, alpha, related sequence 1

AY761184 cDNA sequence AY761184Gm7849 predicted gene 7849Gm7861 predicted gene 7861

167 10429555 2010109I03Rik RIKEN cDNA 2010109I03 gene 3.2696168 10471880 Mir181b-2 microRNA 181b-2 3.2547169 10466110 Ms4a10 membrane-spanning 4-domains, subfamily A, member 10 3.2492170 10444244 Tap1 transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) 3.2423171 10472757 Cybrd1 cytochrome b reductase 1 3.2383

Socs2 suppressor of cytokine signaling 2Gm9847 suppressor of cytokine signaling 2 pseudogene

173 10399478 Lpin1 lipin 1 3.1918174 10463282 Entpd7 ectonucleoside triphosphate diphosphohydrolase 7 3.1653

Ifi47 interferon gamma inducible protein 47Olfr56 olfactory receptor 56

176 10492136 Dclk1 doublecortin-like kinase 1 3.1517177 10538214 D330028D13Rik RIKEN cDNA D330028D13 gene 3.1417178 10456248 Onecut2 one cut domain, family member 2 3.1085179 10459071 2010002N04Rik RIKEN cDNA 2010002N04 gene 3.0889180 10585282 2010007H06Rik RIKEN cDNA 2010007H06 gene 3.0811181 10390860 Krt23 keratin 23 3.0759182 10523547 Agpat9 1-acylglycerol-3-phosphate O-acyltransferase 9 3.0759

Gsta1 glutathione S-transferase, alpha 1 (Ya)Gm10639 predicted gene 10639

Gsta2 glutathione S-transferase, alpha 2 (Yc2)Gm3776 predicted gene 3776Gm1502 predicted gene 1502Gm8760 predicted gene 8760Igkv4-71 immunoglobulin kappa chain variable 4-71

185 10526514 Cldn15 claudin 15 3.0112

No Affimetrix Transcripts Cluster ID Gene Symbol Gene Description Fold Change Graft/Colon1 10497337 Car1 carbonic anhydrase 12 10476042 Tgm3 transglutaminase 3, E polypeptide3 10463005 Cyp2c55 cytochrome P450, family 2, subfamily c, polypeptide 554 10568165 Zg16 zymogen granule protein 16

Saa1 serum amyloid A 1Saa2 serum amyloid A 2

6 10424683 Ly6g lymphocyte antigen 6 complex, locus G7 10519527 Abcb1a ATP-binding cassette, sub-family B (MDR/TAP), member 1A8 10430873 Cyp2d34 cytochrome P450, family 2, subfamily d, polypeptide 349 10599008 Slc6a14 solute carrier family 6 (neurotransmitter transporter), member 1410 10551531 Sycn syncollin

Selenbp1 selenium binding protein 1Selenbp2 selenium binding protein 2

1050350810503523

13 10500570 Hao2 hydroxyacid oxidase 214 10395201 Slc26a3 solute carrier family 26, member 315 10415446 Atp12a ATPase, H+/K+ transporting, nongastric, alpha polypeptide16 10451016 Mep1a meprin 1 alpha17 10547206 Fxyd4 FXYD domain-containing ion transport regulator 418 10411082 Thbs4 thrombospondin 419 10448402 Prss30 protease, serine, 3020 10593198 Fam55b family with sequence similarity 55, member B21 10568001 Sult1a1 sulfotransferase family 1A, phenol-preferring, member 122 10478525 Wfdc2 WAP four-disulfide core domain 223 10500555 Hsd3b3 hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 324 10592266 Slc37a2 solute carrier family 37 (glycerol-3-phosphate transporter), member 225 10557300 Aqp8 aquaporin 826 10514708 Insl5 insulin-like 5

184 10545187

183 10587331 3.0649

11 10494114

12 Ggh

172 10394674 3.2008

3.0407

List of genes enriched in Colon

5 10563611

175 10375515 3.1548

149 10450735 3.5162

166 10577421 3.2753

147 Rgs5 regulator of G-protein signaling 5 3.5231

gamma-glutamyl hydrolase

27 10380614 Hoxb13 homeobox B1328 10531100 Sult1d1 sulfotransferase family 1D, member 129 10585194 Il18 interleukin 1830 10391561 Pyy peptide YY31 10469151 Itih5 inter-alpha (globulin) inhibitor H532 10471464 St6galnac6 ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 633 10365640 Slc5a8 solute carrier family 5 (iodide transporter), member 834 10407792 Gpr137b-ps G protein-coupled receptor 137B, pseudogene35 10377938 Eno3 enolase 3, beta muscle36 10516484 Gjb3 gap junction protein, beta 337 10483074 Gcg glucagon38 10354777 Satb2 special AT-rich sequence binding protein 2

Acsm3 acyl-CoA synthetase medium-chain family member 3Eri2 exoribonuclease 2

40 10419034 2610528A11Rik RIKEN cDNA 2610528A11 gene41 10459183 Slc26a2 solute carrier family 26 (sulfate transporter), member 242 10514912 Dio1 deiodinase, iodothyronine, type I43 10440647 Cldn8 claudin 844 10440655 2310079G19Rik RIKEN cDNA 2310079G19 gene45 10494085 Selenbp2 selenium binding protein 246 10507137 Pdzk1ip1 PDZK1 interacting protein 147 10472946 Hoxd13 homeobox D1348 10359422 Prdx6 peroxiredoxin 649 10574498 Ces2a carboxylesterase 2A50 10598771 Maoa monoamine oxidase A51 10537690 Sval1 seminal vesicle antigen-like 152 10581824 Fa2h fatty acid 2-hydroxylase53 10539156 Gm15401 predicted gene 1540154 10575833 Hsd17b2 hydroxysteroid (17-beta) dehydrogenase 255 10538163 Abp1 amiloride binding protein 1 (amine oxidase, copper-containing)56 10425772 Cyp2d12 cytochrome P450, family 2, subfamily d, polypeptide 1257 10406407 Arrdc3 arrestin domain containing 358 10523451 Anxa3 annexin A359 10430319 Tst thiosulfate sulfurtransferase, mitochondrial60 10394060 Sectm1b secreted and transmembrane 1B61 10373600 Mettl7b methyltransferase like 7B62 10467385 Cyp2c68 cytochrome P450, family 2, subfamily c, polypeptide 6863 10376216 Slc36a1 solute carrier family 36 (proton/amino acid symporter), member 164 10523190 Parm1 prostate androgen-regulated mucin-like protein 165 10530145 Tlr1 toll-like receptor 166 10516490 Gjb5 gap junction protein, beta 567 10475990 Slc20a1 solute carrier family 20, member 168 10403291 Akr1c14 aldo-keto reductase family 1, member C1469 10461934 Trpm6 transient receptor potential cation channel, subfamily M, member 670 10454192 Ttr transthyretin71 10588263 Slco2a1 solute carrier organic anion transporter family, member 2a172 10366546 Cpm carboxypeptidase M

Cyp2c40 cytochrome P450, family 2, subfamily c, polypeptide 40Cyp2c67 cytochrome P450, family 2, subfamily c, polypeptide 67Cyp2c68 cytochrome P450, family 2, subfamily c, polypeptide 68Cyp2c69 cytochrome P450, family 2, subfamily c, polypeptide 69

74 10403312 Akr1c19 aldo-keto reductase family 1, member C1975 10428796 Fbxo32 F-box protein 3276 10466606 Anxa1 annexin A177 10367041 Rdh9 retinol dehydrogenase 9

79 10574023 Mt2 metallothionein 280 10602756 Smpx small muscle protein, X-linked81 10565958 P2ry6 pyrimidinergic receptor P2Y, G-protein coupled, 682 10536273 Casd1 CAS1 domain containing 183 10411373 Hexb hexosaminidase B

Nr1d1 nuclear receptor subfamily 1, group D, member 1Thra thyroid hormone receptor alpha

85 10423333 Fam134b family with sequence similarity 134, member B86 10598203 Ccl28 chemokine (C-C motif) ligand 2887 10574780 Hsd11b2 hydroxysteroid 11-beta dehydrogenase 288 10515282 Tspan1 tetraspanin 189 10433445 Abat 4-aminobutyrate aminotransferase90 10376376 1810065E05Rik RIKEN cDNA 1810065E05 gene91 10574524 Ces2c carboxylesterase 2C92 10514421 Lrrc19 leucine rich repeat containing 1993 10590957 Fut4 fucosyltransferase 494 10349295 Tcfcp2l1 transcription factor CP2-like 195 10572949 Nr3c2 nuclear receptor subfamily 3, group C, member 296 10404059 Hist1h1c histone cluster 1, H1c97 10580765 Pllp plasma membrane proteolipid98 10437191 B3galt5 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 599 10469816 Il1rn interleukin 1 receptor antagonist100 10391555 Ppy pancreatic polypeptide101 10379176 Unc119 unc-119 homolog (C. elegans)102 10496438 Adh1 alcohol dehydrogenase 1 (class I)103 10490923 Car2 carbonic anhydrase 2104 10354374 Slc40a1 solute carrier family 40 (iron-regulated transporter), member 1105 10458704 Spink3 serine peptidase inhibitor, Kazal type 3106 10429564 Ly6a lymphocyte antigen 6 complex, locus A107 10479047 Pck1 phosphoenolpyruvate carboxykinase 1, cytosolic108 10452202 Acer1 alkaline ceramidase 1109 10580286 Best2 bestrophin 2110 10390748 Tns4 tensin 4111 10356968 Pam peptidylglycine alpha-amidating monooxygenase

Eif4ebp3 eukaryotic translation initiation factor 4E binding protein 3Ankhd1 ankyrin repeat and KH domain containing 1

113 10492300 Aadac arylacetamide deacetylase (esterase)114 10501222 Gstm2 glutathione S-transferase, mu 2115 10348062 B3gnt7 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 7116 10422598 Sepp1 selenoprotein P, plasma, 1

112 10454944

glutamate-ammonia ligase (glutamine synthetase)Glul1037445378

39 10556769

73 10467390

84 10390691

117 10606868 Bex1 brain expressed gene 1118 10495675 F3 coagulation factor III119 10472958 Hoxd10 homeobox D10120 10492021 Postn periostin, osteoblast specific factor121 10432652 Cela1 chymotrypsin-like elastase family, member 1122 10375667 Rnf130 ring finger protein 130123 10501229 Gstm1 glutathione S-transferase, mu 1124 10519886 Sema3c sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C125 10589076 Slc25a20 solute carrier family 25 (mitochondrial carnitine/acylcarnitine translocase), member 20126 10601484 2010106E10Rik RIKEN cDNA 2010106E10 gene127 10529819 Fgfbp1 fibroblast growth factor binding protein 1128 10431711 Slc2a13 solute carrier family 2 (facilitated glucose transporter), member 13129 10511870 Fut9 fucosyltransferase 9130 10466130 Ms4a8a membrane-spanning 4-domains, subfamily A, member 8A131 10503259 Trp53inp1 transformation related protein 53 inducible nuclear protein 1132 10388834 Slc13a2 solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 2133 10514221 Plin2 perilipin 2134 10501879 Usp53 ubiquitin specific peptidase 53135 10445347 Clic5 chloride intracellular channel 5136 10504743 Nans N-acetylneuraminic acid synthase (sialic acid synthase)

10348902 Gal3st2 galactose-3-O-sulfotransferase 210348896 Gm6086 predicted gene 6086

138 10422412 Slc15a1 solute carrier family 15 (oligopeptide transporter), member 1139 10493794 S100a14 S100 calcium binding protein A14140 10603746 Maob monoamine oxidase B141 10497296 Slc10a5 solute carrier family 10 (sodium/bile acid cotransporter family), member 5142 10411156 Scamp1 secretory carrier membrane protein 1143 10365482 Timp3 tissue inhibitor of metalloproteinase 3

1036251110501218

145 10461856 Gna14 guanine nucleotide binding protein, alpha 14146 10542872 Rps4y2 ribosomal protein S4, Y-linked 2147 10599736 Fhl1 four and a half LIM domains 1148 10430866 Cyp2d10 cytochrome P450, family 2, subfamily d, polypeptide 10149 10548817 Plbd1 phospholipase B domain containing 1150 10414065 Anxa8 annexin A8151 10474809 Phgr1 proline/histidine/glycine-rich 1152 10497149 Wls wntless homolog (Drosophila)153 10541885 Scnn1a sodium channel, nonvoltage-gated 1 alpha154 10466521 Gcnt1 glucosaminyl (N-acetyl) transferase 1, core 2

Ces2d-ps carboxylesterase 2D, pseudogeneCes2c carboxylesterase 2CCes2b carboxyesterase 2B

156 10357239 Tmem37 transmembrane protein 37157 10436978 Cbr3 carbonyl reductase 3158 10436945 Slc5a3 solute carrier family 5 (inositol transporters), member 3159 10542822 Rep15 RAB15 effector protein160 10482030 Stom stomatin161 10421774 Dgkh diacylglycerol kinase, eta162 10511975 Slc35a1 solute carrier family 35 (CMP-sialic acid transporter), member 1163 10506658 Hspb11 heat shock protein family B (small), member 11164 10426891 Mettl7a1 methyltransferase like 7A1

Gm10035 predicted gene 10035Mettl7a2 methyltransferase like 7A2Mettl7a1 methyltransferase like 7A1Higd1c HIG1 domain family, member 1C

166 10487011 Gatm glycine amidinotransferase (L-arginine:glycine amidinotransferase)167 10538791 Tnip3 TNFAIP3 interacting protein 3168 10437804 Pla2g10 phospholipase A2, group X169 10544982 Nt5c3 5'-nucleotidase, cytosolic III170 10439282 Csta cystatin A171 10413086 Adk adenosine kinase172 10411459 Tmem171 transmembrane protein 171173 10434758 St6gal1 beta galactoside alpha 2,6 sialyltransferase 1174 10469353 Fam23a family with sequence similarity 23, member A175 10575328 Phlpp2 PH domain and leucine rich repeat protein phosphatase 2176 10461979 Aldh1a1 aldehyde dehydrogenase family 1, subfamily A1177 10575184 Wwp2 WW domain containing E3 ubiquitin protein ligase 2178 10494288 Golph3l golgi phosphoprotein 3-like179 10365983 Lum lumican180 10493789 S100a13 S100 calcium binding protein A13181 10572928 Rasd2 RASD family, member 2182 10402473 Clmn calmin183 10556491 Far1 fatty acyl CoA reductase 1184 10400143 Stxbp6 syntaxin binding protein 6 (amisyn)185 10380599 Ttll6 tubulin tyrosine ligase-like family, member 6186 10361828 Cited2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2187 10419578 Ndrg2 N-myc downstream regulated gene 2188 10494761 Vtcn1 V-set domain containing T cell activation inhibitor 1

Tgoln1 trans-golgi network proteinTgoln2 trans-golgi network protein 2

190 10544795 Mir196b microRNA 196b191 10519607 4930420K17Rik RIKEN cDNA 4930420K17 gene192 10365971 Btg1 B-cell translocation gene 1, anti-proliferative193 10415572 2410022M11Rik RIKEN cDNA 2410022M11 gene

Mrps6 mitochondrial ribosomal protein S6Slc5a3 solute carrier family 5 (inositol transporters), member 3

195 10389222 Ccl6 chemokine (C-C motif) ligand 6Tesk2 testis-specific kinase 2Toe1 target of EGR1, member 1 (nuclear)

197 10348906 Gm6086 predicted gene 6086198 10467110 Lipo1 lipase, member O1199 10537545 Gm7254 predicted gene 7254200 10402347 Ifi27l2a interferon, alpha-inducible protein 27 like 2A201 10410351 AA987161202 10475532 Sqrdl sulfide quinone reductase-like (yeast)203 10404359 Mboat1 membrane bound O-acyltransferase domain containing 1

10507347

165 10426894

189 10545450

194 10436941

196

137

144 Gstm3 glutathione S-transferase, mu 3

155 10574532

204 10502732 Prkacb protein kinase, cAMP dependent, catalytic, beta205 10434302 Klhl24206 10481435 Ccbl1 cysteine conjugate-beta lyase 1207 10477169 Id1 inhibitor of DNA binding 1

Klk1 kallikrein 1Klk1b5 kallikrein 1-related peptidase b5Klk1b4 kallikrein 1-related pepidase b4

209 10480145 Rsu1 Ras suppressor protein 1

208 10552546

supplemental material small intestinal stem cell identity...

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