DOI: 10.1038/ncb2610 Figure S1 FSMCs derived from MSLN CLN transgenic mice express smooth muscle-specific proteins. Beta-galactosidase is ubiquitously expressed within cultured FSMCs derived from MSLN CLN transgenic mice (a-a ). FSMCs derived from cultured mesothelium express Smooth Muscle-Myosin Heavy Chain II (b-b ) and Smooth Muscle-Myosin Heavy Chain XI (c-c ). Scale bars= 100µm (a-a ), 50µm (b-b, c-c ). WWW.NATURE.COM/NATURECELLBIOLOGY 1
Figure S2 Mesothelin (MSLN) protein is expressed on the mesothelium covering the visceral and parietal tissues and organs. MSLN is ubiquitously expressed within the mesothelial tissue surrounding the bladder (a-a ), intestine (b-b ), heart (c-c ), stomach (d-d ), spleen (e-e ), liver (f-f ), lung (g-g ), mesentery (h-h ), thymus (i-i ) and peritoneum (j-j ). Nuclear DAPI staining (a-j), MSLN staining (a -j ), merged images [MSLN staining in green or red, nuclear DAPI staining in blue or white] (a -j ). Scale bars= 200µm (a-j). 2 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S3 Boolean relationships of MSLN. Relationship was inferred by using a Boolean algorithm on 10,823 Affymetrix 430 2.0 mouse arrays that were downloaded from NCBI s Gene Expression Omnibus (a) Each dot represents a boolean relationship of Mesothelin (MSLN) on a single Affymetrix 430 2.0 mouse array from NCBI s Gene Expression Omnibus showing all tissues in red, and adult mouse lung in blue. X-axis represents Mesothelin expression within the specific arrays, Y-axis represents the expression of each specific other marker. A high-to-high Boolean relationship exists between MSLN and Type 4A1 Collagen (Col4a1), Type 4A2 Collagen (Col4a2), Type 5A2 Collagen (Col5a2), Fibronectin 1 (Fn1), alpha2-smooth muscle actin-aorta (Acta2), alpha1-smooth muscle actinin (Actn1)and Smoothelin (Smtn) and Vimentin (Vim). Light blue dots represent boolean relationships of MSLN on arrays from adult mouse lung epithelium and cancer cell lines, which most likely are separate from the FSMC lineage. (b) Adult mouse airway epithelium express MSLN protein, separate from the FSMC lineage. Nuclear DAPI staining (b), MSLN staining (b ), merged image [MSLN in green, nuclear DAPI staining in blue] (b ). Scale bars=100µm. WWW.NATURE.COM/NATURECELLBIOLOGY 3
Figure S4 Developmental restrictions of MSLN + precursors to FSMCs during adult organ maintenance. Sections through representative organs, 4-months following tamoxifen administration. Stomach (a-a ), intestine (b-b ), liver (c-c ), thymus (d-d ), heart atrium (e-e ). Nuclear DAPI staining (a-e), betagalactosidase staining (a -e ), merged images [beta-galactosidase staining in green, nuclear DAPI staining in grey]. Co-localization of beta-galactosidase with αsma protein within the intestine (f-f ) and stomach (g-g ) smooth muscle layers. Beta-galactosidase staining (f, g), αsma staining (f, g ), merged images [beta-galactosidase staining in green, αsma staining in red, nuclear DAPI staining in grey] (f, g ). Scale bars= 100µm (a-d, f, g), 200µm (e). 4 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S5 Negligable differentiation to FSMCs in the trunk vasculature following whole-body irradiation. Sections through trunk organs from MSLN CLN ;R26 mtmg transgenic mice showing absence of beta-galactosidase staining within trunk vasculature, 1-month following whole-body irradiation. Mesenteric vasculature (a-a ), pulmonary vasculature (b-b ), pulmonary artery (c-c ), coronary vasculature (d-d ), white arrowheads. Nuclear DAPI staining (a-d), beta-galactosidase staining (a -d ), mt (a -d ), merged images [mt in red, DAPI in white] (a -d ). Scale bars= 100µm (a, b, d), 200µm (c). WWW.NATURE.COM/NATURECELLBIOLOGY 5
Figure S6 Minimal contributions from neural crest to trunk FSMCs. (a-g) Merged images from Wnt1 Cre R26 mtmg transgenic mice. GFP expression is restricted to peripheral nerves innervating the smooth muscle and trunk vasculature. Sections through the lower digestive system (a, b), urinary bladder (c), lungs (d), coronary vasculature (e), renal vasculature (f) and mesenteric vasculature (g). Nuclear DAPI staining in gray, mg in green, αsma in red. (h-j) Wnt1 Cre R26 mtmg -lineage derived neural crest express β3-tubulin invivo. (h) urinary bladder (i) stomach (j) intestine. Nuclear DAPI staining in gray, mg in green, β3-tubulin in purple. (k-o) Negligible contributions from neural crest to FSMCs in-vitro. Merged images of mg (green) and mt (red) of mesothelium cultured from adult Wnt1 Cre R26 mtmg mice. (k) intestine (l) stomach (m) urinary bladder (n) liver (o) and heart, showing absence of GFP within cultured FSMCs. (p-u) Absence of contributions in-vivo to FSMCs from Sox9 Cre R26 mtmg -lineage derived neural crest. (p, q) Sections through the intestine (smooth muscle layer is outlined by dotted line), (r) urinary bladder (smooth muscle layer is outlined by dotted line), (s) heart ventricle (coronary vasculature is outlined by dotted line), (t) gut vasculature (outlined by dotted line) and lung vasculature (u). GFP is absent within the smooth muscle layers of the internal organs and their vasculature. Scale bars=original magnifications: 200µm (p), 100µm (a-j, q-u), 50µm (k-o), 200µm (f, u). 6 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S7 Negligible contributions in-vivo to FSMCs from Mpz Cre R26 lacz - lineage derived neural crest. Eosin staining of sections through the intestine (a-a ), kidney (b-b ), liver (c-c ) and cardiac vasculature (d-d ) from Mpz Cre ;R26 lacz transgenic mice. LacZ staining is present within neural plexus in the smooth muscle layers of the digestive system (white arrows in a, a ) and its vasculature (outlined by dotted lines in a, a ). LacZ is absent from the intestinal, renal, hepatic and cardiac vasculatures. LacZ staining is present within the aortic arch of the heart (white arrows in d, dotted lines in d ). Eosin staining in red, lacz staining in dark blue. Scale bars=100µm. WWW.NATURE.COM/NATURECELLBIOLOGY 7
Figure S8 Minimal contributions of circulating cells to trunk FSMCs throughout adulthood. Images of the mouse abdominal cavity following 1 year of parabiosis. Small intestine (a, a ), large intestine (b, b ), mesentery (c, c ) and lymph nodes (d, d ). Bright field images (a-d), fluorescent images (a -d ). Sections of representative organs from parabiosed mice (e-i). Duodenum (e), kidney (f), urinary bladder (g), lung (h) and liver (i). Nuclear DAPI staining (e-i), GFP expression (e -i ), CD45 expression (e - i ), merged images [GFP in green, antibody staining in red, nuclear DAPI staining in blue] (e -i ). Scale bars= 1mm (a-c), 100µm (e-i), 300µm (b, d). 8 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Movies Movie S1 Time-lapse video (72 hours) of a cultured explant from the mesothelium overlying the intestine, two days post-culture. Multiple FSMCs continuously emerge from the periphery of the explant concomitant with shrinkage of the explant size. Emerging FSMCs display a spindle-shape or a flattened morphology and are highly motile within the culture dish, continuously forming filopodia and lamelipodia in the direction of their migration. Movie S2 Time-laps video (72 hours) of a cultured explant from the mesothelium overlying the kidney, four days post-culture. Contractile forces from newly emerging FSMCs at leading edge sites of the tissue, lead to pulling of the tissue explant along the culture plate. Movie S3 Time-laps video (72 hours) of a cultured explant from the mesothelium overlying the mesentery, seven days post-culture. Emerging FSMCs do not exhibit directed movement, but rather sample the tissue culture plate, continuously changing their direction of cell migration. WWW.NATURE.COM/NATURECELLBIOLOGY 9
Supporting online materials Supplementary Table 1 A list of accession numbers of mouse arrays from NCBI s Gene Expression Omnibus (GEO) that represent transcripts with a high-to-high Boolean relationship with Mesothelin (MSLN). We downloaded publicly available Affymetrix Mouse 430 2.0 mouse microarrays from GEO and normalized them all together using RMA (Robust Multichip Average) algorithm. StepMiner algorithm and BooleanNet statistics were used to determine thresholds for each probeset and to create Boolean implication relationships 46.