Moore-Morris et al. Supplemental Table 1.

Transcription

1 Moore-Morris et al. Supplemental Table. In vivo echocardiographic assessment of cardiac size and function following transaortic constriction (T) at 7d and 8d. SHM 7d N=6 T 7d N=5 SHM 8d N= T 8d N=6 W, g.± ±.8.6± ±.5 HR, bpm 57±7 5±5 55±7 59±5 IVSd, mm.7±.9.±.7*.67±.5.9±.* LVIDd, mm.67±.6.59±..55±.5.±.5* LVIDs, mm.±..9±..7±.7.9±.8* LVFS 6.9± ±6.78.±5..6±9.9* TSPG, mmhg N/ 8.±5.5 N/ 86.±6.5 HR, heart rate; LVIDd, LV internal dimension at end-diastole; LVIDs, LV internal dimension at end-systole; TSPG, trans-stenotic systolic pressure gradient as the difference between right carotid and left axillary artery systolic pressures. Results are expressed as mean ± SD. *P <.5 by Student s t-test before and after T within the same group.

2 Moore-Morris et al. Supplemental Figure LV/W (mg/g) 6 5 p<. p<. 7d T 8d T T T Trichrome staining 7d 8d anp fold change p=. p<. p=. ßmhc fold change 8 6 p=.9 p=.9 p=. 5 5 cola fold change p=. p=.6 p=. p<. cola fold change p<. p=.9 αmhc fold change p=. p=.9 p=.5 7d T 8 d T Supplemental Figure. Transaortic constriction (T) model. () Left ventricle (LV) weight over body weight (W). Data was analyzed by Student s t-test (n=6-8 per group). () Representative trichrome staining of left ventricular wall. () qrt-pr analysis of genes associated with hypertrophy/fibrosis. Data was analyzed by unpaired Student s t-test. Scale bars represent µm.

3 Moore-Morris et al. Supplemental Figure % cells in perivascular fibrotic lesion 7d T 6 5 olagfp+;fsp- olagfp+;fsp+ FSP+;olaGFP-;- FSP+;+ +;FSP- DPI FSP DPI FSP D % cells in perivacsular fibrotic lesion 7d T 6 5 olagfp+;αsm+ olagfp+;αsm- αsm+;olgfp-;- αsm+;+ +;αsm- E DPI αsm F % cells in interstitial fibrotic lesion 8d T 5 olagfp+;αsm+ olagfp+;αsm- αsm+;olgfp-;- αsm+;+ +;αsm- Supplemental Figure. Quantification of cell types in fibrotic areas. () Percentages of cells expressing, FSP and in perivascular fibrotic areas. () onfocal image showing FSP+ fibroblasts (arrows) and an FSP+ leukocyte (arrowhead) in a perivascular area. () onfocal image showing FSP labels immune cells (+) and not perivascular fibroblasts in operated mice. (D) Percentages of cells expressing, αsm and in perivascular fibrotic areas. (E) onfocal image showing lack of αsm + fibroblasts in a perivascular area. No ollagenagfp+ cells were +. (F) Percentages of cells expressing, αsm and in interstitial lesions following 8d T. ells were counted in fields each from mice. Histograms represent mean±s.d. Scale bars are µm., coronary artery.

4 Moore-Morris et al. Supplemental Figure trl 7d T % % % % 8d T % % PDGFRα.7% 9.5%.5% 5.%.%.% 7d T 8d T vimentin DPI ollagena -GFP merge Fold change compared with endothelium Fold change compared with endothelium 5 5 ddr prolyl--hydroxylase d T 7d T Supplemental Figure. Expression of fibroblast markers by + cells () Representative flow cytometry analysis showing that collagena-gfp+ cells are PDGFRa+ in operated animals and following 7d and 8d T. ll plots/images are representative of at least 5 animals per group. () Represesentative images of vimentin staining in operated animals and following 7d and 8d T. Scale bars represent µm. () qrt-pr data showing expression level of DDR and Prolyl--hydroxylase in + cells versus + endothelial cells sorted from (n=) and 7d T (n=) hearts.

5 Moore-Morris et al. Supplemental Figure dult (5w) WT-cre;Rosa-tdT DPI image of atria and RV * * * * Left atrium Right Ventricle E8.5 Tbx8-cre;Rosa-tdT DPI b b b b IVS E8.5 Tbx8-cre;Rosa-tdT DPI c c c LVFW c Supplemental Figure. Distribution of fibroblasts derived from epicardium. () onfocal images of adult Wt-cre lineage traced atria and right ventricle. Fibroblasts were lineage-traced (arrows). (*) Epicardial surface. () onfocal images of E8.5 Tbx8-cre lineage-traced hearts. s seen with Wt-cre, many fibrblasts were ot labelled in the interventricular septum (IVS) (arrows). () vast majority of fibroblasts were labelled in left ventricular free wall (LVFW) of Tbx8-cre lineage-traced hearts (arrows).scale bars represent 5µm.

6 Moore-Morris et al. Supplemental Figure 5 LVFW IVS DPI VEcadherine-creERT;Rosa-tdT Tie-cre;Rosa-tdT D RV DPI Tie-cre;Rosa-tdT RV DPI Wt-cre;Rosa-tdT E 7d T DPI Tie Supplemental Figure 5. Lack of EndoMT following pressure overload. () Perivascular fibrosis in Tiecre+/-;ollagenaGFP+/;RosaTdt+/- hearts. Following 7d T, as observed for intersitital fibroblasts, perivascular fibroblasts in left ventricular free wall (LVFW) were Tiecre-RosatdT- whereas perivascular fibroblasts in interventricular septum (IVS) were Tiecre-RosatdT+. Similar results were observed at 8d T. Scale bars represent 5mm. () Perivascular fibrosis in VE-cadherin lineage traced heart following 7d T. + coronary endothelial cells were labelled, but not perivascular fibroblasts. Scale bar mm. () Right ventricle (7d T) from Tie-cre lineage traced heart. (D) Equivalent area to inset in () in RV of Wt-creERT lineage traced heart. Similar results were observed at 8d T. Scale bars represent 5µm. (E) ells expressing Tie protein (arrows) were not + fibroblasts in hearts and following 7d T. Similar results were observed at 8d T. Scale bars represent µm.

7 Moore-Morris et al. Supplemental Figure 6 Tie-cre SHM F Singlets Rosa-tdT Singlets Rosa-tdT 86.%.%.8%.8% 6.% 9.% 89.6%.%.8% Tie-cre 8d T 9.6%.8% 5.%.%.5%.9%.% D E Singlets SHM Rosa-tdT.6% 7.% 7.5% 5.6%.9% IVS Tie-cre 7d T.%.%.% Tie-cre;Wt-cre 8d T 9.6% of GFP+ Rosa-tdT Tie-cre;Rosa-tdT DPI Edu LVFW d T 6.% of GFP+ DPI leaved-caspase- Supplemental Figure 6. Similar responses of Tie-re and non Tie-re derived fibroblasts to pressure overload () Flow cytometry plot of dissociated left ventricle (LV) + interventricular septum (IVS) from a sham operated Tiecre+/-;collagena-GFP+/-;Rosa-tdT+/- mouse. Tie-cre;Rosa-tdT+, Tie-cre;Rosa-tdT++ and Tie-cre;Rosa-tdT+- populations have been gated and plotted against and expression. oth + populations are negative for and, except a small subset of collagena-gfp+;+ endocardial cells. Tie-cre;Rosa-tdT+;collagena-GFP- cells consist of + endothelial cells and + leukocytes. This is also the case following 7d () and 8d () T. (D) Representative flow cytometry plots showing percentages of labelled (Rosa-tdT+) and non-labelled (Rosa-tdT-) Fs in double Tie- cre;wt-cre lineage traced LV + IVS (representative of mice). (E) Representative images of IVS and LVFW (left ventricular free wall) of sham operated and T operated Tie-cre+/-;collagena-GFP+/-;Rosa-tdT+/- mice d after surgery (PG 76.7±8. mmhg, n=). Hearts were recovered h after EdU injection. Rare EdU+ cells (arrowheads) were never fibroblasts in hearts. Equivalent fibrotic areas that contained EdU+ fibroblasts (arrows) were found in IVS and LVFW. Scale bars are µm. (F) poptotic leaved-caspase + cells were very rare following T and - (see inset). The reporter is cytoplasmic and nuclear, enabling clear identification of fibroblast nuclei (see nucleus to right of aspase + cell inset). Scale bars are µm except insets (5µm). Image taken from 7d post surgery, similar results were observed following d and 8d T.

8 Moore-Morris et al. Supplemental Figure 7. Vav-cre;Rosa-tdT+ gate LV+ IVS Singlets - Vav-cre;Rosa-tdT+ gate 7d T 8d T - - Supplemental Figure 7. Vav-cre labelling following pressure overload. () Representative flow cytometry plots showing Vav-cre;Rosa-tdT+ and signals from cell suspensions from LVFW+IVS. >9% of + leukocytes were labelled in Vav-cre lineage traced mice, with almost no labelling outside of this cellular compartment. () Vav-cre lineage traced cells consist invariably of + leukocytes in and following 7d or 8dT, with small sub-populations of + endothelial cell. Each plot is representative of at least mice.

9 Moore-Morris et al. Supplemental Figure 8 ge (weeks): nalysis nalysis SHM/T surgery Tamoxifen inductions (5 consecutive days) Recombination efficiency in endothelium Rosa-tdT Singlets 5.7% - 8.8% Supplemental Figure 8. Inductions in VE-cadherin-reERT lineage traced mice () Time line for tamoxifen induction and T procedure on VE-cadherin-reERT+/-;collagena-GFP+/-;Rosa- Tdt+/- mice. () Representative flow cytometry plot showing the percentage of + endothelium labeled in left ventricle (LV) + interventicular septum (IVS) by VEcadherin-creERT following inductions.

10 Moore-Morris et al. Supplemental Figure 9 ge (weeks): nalysis /T surgery Tamoxifen inductions ( consecutive days) d T Tbx8-reERT-RosatdT DPI PDGFRα D6 Supplemental Figure 9 Epicardium does not give rise to fibroblasts following pressure overload. () Time course for tamoxifen inductions and /T surgery for Tbx8-reERT+/-;Rosa-tdT+/- mice. () onfocal images showing recombination in epicardium (asterisks) and D6+ mural cells (arrowheads) but not in PDGFRα+ fibroblasts (arrows) of operated mice and following d pressure overload (PG 8±.5 mmhg, n=). Scale bars represent µm.

11 Moore-Morris et al. Supplemental Figure Embryonic DPI αsm PDGFRα dult DPI αsm DPI E5.5 Nfatc-cre;Rosa-tdT L PDGFRα PDGFRα DPI LVFW D E5.5 Nfatc-cre;Rosa-tdT DPI IVS Supplemental Figure. Lack of PDGFRα staining in vascular smooth muscle/epicardium and confocal images of E5.5 IVS of Nfatc-cre+/-;+/-;Rosa-tdT+/- embyronic heart. () Embryonic E7.5 left ventricular wall. Epicardium is indicated by arrows. () dult smooth muscle (5 weeks old male) is PDGFRα, wherease epicardial cells (arrows) express PDGFRα. () LVFW displaying extensive labelling of + vessels (arrows). (D) s with Tie-re and VE-cadherin-creERT, Nfatc-cre lineage traced fibroblasts were abundant in IVS (arrows). L, left atrium. Scale bars in and represent µm, Scale bars in and D represent 5µm.

12 Moore-Morris et al. Supplemental Figure VE-cadherin-creERT;Rosa-tdT E7.5>E5.5 DPI VE-cadherin-creERT;Rosa-tdT E9.5>E5.5 DPI VE-cadherin-reERT;Rosa-tdT E7.5>E9.5 D VE-cadherin-reERT;Rosa-tdT E7.5>E.5 E OFT R E E RV E VEcadherin Wt DPI VEcadherin Wt Supplemental Figure. Fate of VE-cadherin-reERT lineage traced endocardium. () Tamoxifen induction at E7.5 in VE-cadherin-creERT embryos resulted in labelling of + fibroblasts in the valves and septum (&), and labelling of a small number of fibroblasts in the ventricular wall by E5.5 (, arrows). () Tamoxifen induction at E9.5 resulted in no labelling of fibroblasts, despite a similar degree of labelling of endothelium. () Tamoxifen induction in E7.5 embryos resulted in Rosa-tdT+/- lineage labeling of VE-cadherin immunolabeled endothelial/endocardial cells but not Wt immunolabeled proepicardial cells at E9.5. (D) Inductions at E7.5 and harvesting at E.5 resulted in labeling of atrioventriclar canal cushion (e ) and VE-cadherin+ endocardial/endothelial cells, but not Wt+ epicardial cells (e ). Scale bars are µm except chamber views in () and () that represent µm. OFT= Outflow tract, R= Right atrium.

13 Moore-Morris et al. Supplemental Figure Fold difference - qrt-pr. postn wisp pdgfrα ddr cola. Fold difference - microarray R =.8 cdh5 (VEcad) pecam cola luster GO Term Gene number Permute P value T EFFET extracellular matrix part(go:) <. FIROLSTS proteinaceous extracellular 7 <. (luster ) matrix(go:5578) metallopeptidase activity(go:87) <. negative regulation of cell death(go:658) 9 <. positive regulation of fibroblast proliferation (GO:86) <. FIROLST SPEIFI (luster ) ENDOTHELIL ELL SPEIFI (luster ) T EFFET FIROLSTS ND ENDOTHELIL ELLS (luster ) extracellular matrix(go:) <. regulation of cartilage 7 <. development(go:65) pattern binding(go:87) 65 <. extracellular matrix part(go:) 65 <. extracellular matrix organization(go:98) 5 <. adherens junction(go:59) 6.5 regulation of small GTPase mediated signal 6.85 transduction(go:556) cell-cell adherens junction(go:59). cadherin binding(go:596).5 regulation of angiogenesis(go:5765).55 cell cycle(go:79) 7 <. cell cycle process(go:) 6 <. cell division(go:5) 9 <. chromosome, centromeric region(go:775) 5 <. chromosome(go:569) 79 <. Supplemental Figure. Genes differentially regulated following pressure overload. () Plot showing correlation between q-rt- PR and microarray fold differences between genes from clusters (T effect on fibroblasts), (fibroblast specific genes) and (endothelial specific genes) of Figure 9. Genes from cluster are cola (D/) and wisp (/). Genes from cluster are cola (/E), ddr (/E), pdgfrα (D/F) and postn (D/). Genes from cluster are cdh5 (VEcadherin) (F/) and pecam (E/) (, TiecreRosatdT+ fibroblasts ;, TiecreRosatdT- fibroblasts ;, TiecreRosatdT+ fibroblasts T; D, TiecreRosatdT- fibroblasts T; E, endothelial cells ; F, endothelial cells T). () Most significant GO-Terms associated with the main gene clusters. () Gene network diagram including genes specifically upregulated in both fibroblast populations following T.