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1 DOI: /nc2824 Hcn4 Tx5 Mlc2 c Hcn4- ISH d Tx5- ISH e Mlc2-ISH Hcn4-ISH f e Tx5-ISH f -ISH Figure S1 Section in situ hyridistion nlysis of crescent stge mouse emryos (E7.5). () More nterior section level of the sme emryo s shown in Figure 1h (~4ss). Expression domins of Hcn4, Tx5 nd Mlc2 (ll shown in green) re predominntly overlpping, ut not with tht of (shown in red). () Corresponding section levels through n E7.5 emryo s shown elow in (c+d). (c-d) Doule in situ hyridistions. Blue stining: NBT/BCIP (BM Purple) nd rown stining: INT/BCIP. (c) Hcn4 (lue) nd (rown) re expressed in predominntly non-overlpping expression domins. (d) Tx5 (lue) nd (rown) re expressed in predominntly non-overlpping expression domins. Expression domins of Hcn4 nd Tx5 re mostly overlpping, when compring their expression on djcent sections. (e) Single fluorescence in situ hyridistions on djcent sections of n E7.5 (~4ss) emryo. (e-f) Expression of Mlc2, Hcn4 nd Tx5 (ll in green) re predominntly expressed within the presumptive FHF, in contrry to expression primrily in the SHF (red). (e -f ) Section levels corresponding to (e) nd (f), respectively. Scle r: 50µm. 1
2 Hcn4-ISH Tx5-ISH -ISH Tx5-ISH -IHC Tx5-ISH -IHC Tx5-ISH -IHC Figure S2 Section in situ hyridistion nd IHC nlysis of crescent stge emryo (E7.5). () Single in situ hyridistions (NBT/BCIP, BM Purple) on djcent sections of n E7.5 (~2-4ss) mouse emryo. Corresponding section levels re shown in pnel on left side. Expression domins of Hcn4 nd Tx5 re mostly overlpping in presumptive FHF, ut not with. However, in the posterior re of the FHF nd SHF domins there is n overlpping expression domin etween Tx5 nd visile (ornge rrows), ut not with Hcn4 (lck rrow), suggesting tht Hcn4 is more exclusive to the FHF thn Tx5. () Co-stining showing single fluorescent in situ hyridistion of Tx5 (green) nd ISL1 protein loction (red) on djcent sections of n E7.5 (~2-4ss) mouse emryo. The two domins re lrgely non-overlpping; however, there is some colocliztion visile in the most lterl sides of the splnchnic mesoderm (rrows). There re lso some res, where there is wek stining for ISL1 protein visile (sterix), overlpping with Tx5 mrna, ut the ISL1 protein does not co-loclize with the nucleus ut seems to e present within the cytoplsm, indicting tht it my not e functionl in these res. Scle r: 50µm. 2
3 -ISH Hcn4-ISH Tx5-ISH Nkx2.5-ISH Mlc2-ISH -ISH Hcn4-ISH Tx5-ISH Mlc2-ISH Figure S3 Section in situ hyridistion nlysis of n erly hert tue stge emryo (E8.0). () Single colour in situ hyridistions on djcent sections. Corresponding section level s shown in drwing. Hcn4, Tx5, Mlc2 nd Nkx2.5 re expressed within the primitive hert tue, unlike, which is expressed in the phryngel mesoderm. At this developmentl stge, Nkx2.5 is mostly non-overlpping with expression. () Single fluorescent in situ hyridistions on djcent sections. Corresponding section levels re shown in drwings ove imges. Expression domins of Hcn4, Tx5 nd Mlc2 (ll green) re mostly overlpping, ut predominntly non-overlpping with expression (red). However, posterior to the primitive hert tue, there is n re of overlp etween Tx5 nd expression (rrow), ut not with Hcn4 nd Mlc2 (most likely no overlp with, s the section level is it more nterior compred to (djcent sections) nd it seems more lterl compred to ). Scle r: 50µm. 3
4 Hcn4 CreErt2/+ ;R26R lcz Tmoxifen: E8.0 Hcn4 CreErt2/+ ;R26R eyfp Tmoxifen: E7.0 c Hcn4 CreErt2/+ ;R26R lcz Tmoxifen: E7.0 dorsl ventrl d e f Hcn4 CreErt2/+ ;R26R lcz Tmoxifen: E7.0 E19.5 E19.5 E19.5 Hcn4 CreErt2/+ ;R26R lcz Tmoxifen: E7.0 ISH E12.5 ventrl -ISH dorsl g Tx5-ISH Hcn4-ISH E10.5 E9.5 E10.5 E9.5 HCN4 CreErt2/+ ;R26R eyfp ; Tmoxifen: E7.0; E19.5 Atri h m-tnnt2- Alex647 Mlc2-ISH tdtomto+ 14% tdtomto+ 12.7% tdtomto+ 14.7% tomto+/tnnt2 82% YFP TNNT2 mgnified HCN4 CreErt2/+ ;R26R eyfp ; Tmoxifen: E7.0; E19.5 Atri PECAM-1-APC tomto+/ PECAM-1 0.1% r-smmhc-alex647 tomto+/ smmhc 1.2% FITC-A FITC-A FITC-A Figure S4 Hcn4+/FHF linege trcing in the emryonic hert nd Hcn4+/ FHF cell linege contriution to tri () Whole-mount X-gl stining of Hcn4 CreErt2/+ ;R26R lcz herts t E19.5. Tmoxifen induced Hcn4 CreErt2/+ ctivtion t E8.0 lels the Hcn4+/FHF derivtives, such s the left ventricle (LV) nd prts of the tri. At this stge, lso the sinus venosus (SV) nd sinotril node (SAN) re stined positive. () Section through n E19.5 Hcn4 CreErt2/+ ;R26R eyfp hert; Hcn4 CreErt2/+ ctivtion y tmoxifen t E7.0. Within the left ventricle, there is Hcn4+/FHF linege contriution to the treculr nd compct lyer of the left ventricle, nd some minor contriution to the septum. (c-d) Whole-mount X-gl stining of Hcn4 CreErt2/+ ;R26R lcz hert t E12.5 (c), nd emryos t E9.5 (d), nd E10.5 (e). Tmoxifen induced Hcn4 CreErt2/+ ctivtion t E7.0 lels the Hcn4+/FHF derivtives, such s the left ventricle (LV) nd prts of the tri. No stining is visile within the phryngel mesoderm (rrows, d+e). In situ hyridistion for of E9.5 emryo mrks the phryngel mesoderm (rrow, f). Hcn4+/FHF cells lso contriute to the AV cnl (red sterix, d+e). (g) Section through n E19.5 tri of Hcn4 CreErt2/+ ;R26R eyfp mice; Hcn4 CreErt2/+ ctivtion y tmoxifen t E7.0. Co-immunostining for YFP s mrker for Hcn4+/FHF linege trced cells nd TNNT2 shows contriution to crdiomyocytes. Yellow ox represents mgnified re shown in merge imge (h) FACS quntifiction of the cellulr contriution of Hcn4+/FHF linege trced cells to n Hcn4 CreErt2/+ ;R26R tdtomto E19.5 tri (Tmoxifen E7.0). FACS nlysis of tomto+ Hcn4+/FHF linege trced cells co-stined for TNNT2, PECAM-1 (CD31) or smmhc (MYH11) showed predominnt contriution to crdiomyocytes, ut no doule positive cells for endothelil/endocrdil-, nd vsculr smooth muscle mrkers, suggesting no contriution to these cell lineges within tri. Representtive FACS dot-plots re shown. Secondry ntiody controls re shown in Suppl. Fig. S7. Scle r: (+c) 0.5mm, () 100µm, (d-f) 0.2mm, (g) 50µm. 4
5 Hcn4 CreErt2/+ ;R26R eyfp ; no Tmoxifen EB dy7 dpi PE A-empty GFP-A yfp+ 0.0% Hcn4 CreErt2/+ ;R26R eyfp ;Tmoxifen EB dy 4+6 PE A-empty EB dy7 dpi yfp+ 2.1% c d Hcn4 Tx5 Nxk2.5 Tnnt2 Mlc2v Cx40 Srcolipin et-ctin Hcn4 CreErt2/+ ;R26R eyfp TNNT H2O H2O Hcn4 CreErt2/+ ;R26R eyfp TNNT2 0mV 0mV 0mV 50mV e Right Ventricle GFP-A 200ms ventriculr-like AP 64% TgAHF-Mef2c-eGFP; E19.5 tril-like AP 16% nodl-like AP 20% % m-tnnt2- Alex % r-smmhc-apc 1.8% 0.4% 29.6 PECAM-1-APC 25.3 Outflow trct, no tri m-tnnt2- Alex % 1.1% 13.2 r-smmhc-apc % 14.0 PECAM-1-APC 3.2% 1.0% 9.8% Figure S5 Clonl nlysis of Hcn4+/FHF cells isolted from mouse ESCs nd TgMef2c-AHF-eGFP/SHF cell linege contriution in vivo. () Emryonic stem cells (ECSs) were derived from Hcn4 CreErt2/+ ;R26R eyfp mice. Mouse ESCs were differentited using the emryoid ody technique. Hcn4 CreErt2/+ ctivity ws induced y dministrtion of 4-OH-Tmoxifen on dy 4 nd 6 of differentition. eyfp positive Hcn4+/FHF cells were FACS sorted (representtive FACS dot-plots re shown). () Single eyfp positive Hcn4+/FHF cells were sorted s 1 cell/well into 96-well pltes. After 2-3 weeks single cell derived clones showed limited expnsion nd efficient differentition into crdiomyogenic clones, visulized y direct fluorescence of eyfp nd immunostining for the crdic muscle mrker TNNT2 (ctnt). (c) RT-PCR nlysis, showing expression of crdic mrker genes in single Hcn4+/FHF cell derived crdiomyogenic clones. (d) Single cell ptch clmp nlysis (n=39). Most clmped cells showed ventriculr-like ction potentil (AP) (64%), while some showed more tril-like AP (16%), or nodl-like AP (20%). Representtive ction potentils re shown. (e) FACS quntifiction of the cellulr contriution of TgMef2c-AHF-eGFP/SHF linege trced cells to the right ventricle nd outflowtrct (OFT). Fixed single cells were co-stined for TNNT2, demonstrting contriution to crdiomyogenic cell lineges, prticulrly within the RV. Co-stining for smmhc (MYH11) showed minor contriution to vsculr smooth muscle cells in RV nd contriution in the OFT. Co-stining for PECAM-1 (CD31) showed minor contriution to endothelil cell linege in RV nd contriution in the OFT. Representtive FACS dot-plots re shown. Scle r: 50µm. 5
6 Hcn4 Hcn4 Nkx2.5 Cx40 Cx Tx5 Mlc2v ctnnt2 Srcolipin et-ctin ckground signl of secondry ntiodies single cells 2.8% single cells single cells 2.1% APC - empty g--m-alex647 APC - empty g--r-alex % 0.2% g--m-alex488 FITC - empty g--r-alex488 FITC - empty Figure S6 Uncropped key electrophoresis dt nd secondry ntiody controls of FACS nlyses. () Uncropped electrophoresis dt of RT-PCR shown in Figure 4c. () Bckground signl of secondry ntiodies used for FACS sorting s shown in Fig. 3f nd Suppl. Fig. 4h. 6
7 Video S1 Clonl nlysis of single Hcn4+/FHF cells isolted from mouse emryos. Representtive exmple of eting crdiomyogenic clone derived from single Hcn4+/FHF cell from mouse emryos. 7
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