SUPPLEMENTARY INFORMATION

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Noah Ramsey
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DOI:./nc In the formt provie y the uthors n uneite.

AVLGIAFGGFLIGVLLTGALWFIKIRTGHPVALGMRSTAAELSVLSISGCPCGLTKRQPV--PTHPSPSENSSANASIGSTQSTP--TSSMA HUMAN AVLGITFGAFLIGALLTAALWYIYSHT------------------------RSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA ** ** *

finch mouse Eng Xenopus / um DMSO BCA PCS PCS PHBC BA PHBC DMSO CtAs DMSO DMSO e / PCS PHBC CtAs um Nifeipine BCA PCS BA PHBC Nifeipine Nifeipine Nifeipine Supplementry Figure Ientifiction of zerfish

() Schemtic representtion of the enoglin locus showing conservtion of gene orer (synteny) etween humn chromosome q- n two prlogous loci on zerfish chromosome contining the Pregnncy-ssocite plsm

Prlogous genes tht were likely uplicte uring the teleost whole genome upliction (WGD) event re inicte in lue.

Dshe lines inicte synteny reks etween humn n zerfish. () Alignment of the conserve C-terminl region of zerfish n humn Enoglin. Asterisks inicte conserve resiues.

1 DOI:./nc In the formt provie y the uthors n uneite. c common fish/humn ncestor teleost WGD Trnsmemrne region PAPPA ppp ppp ASTN stn stn myl-rt* myl-rt* Serine/Threonine-Rich C-terminl omin ZEBRAFISH AVLGIAFGGFLIGVLLTGALWFIKIRTGHPVALGMRSTAAELSVLSISGCPCGLTKRQPV--PTHPSPSENSSANASIGSTQSTP--TSSMA HUMAN AVLGITFGAFLIGALLTAALWYIYSHT RSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA ** ** * * * ** ** * ** ** * ** DBC c c AK k k GPSM gpsm gpsm humn Chr.q- zerfish prlog Chr..-. M zerfish prlog Chr..-. M PDZ-ining motif zerfish tgfr frog tgfr stickleck tgfr fugu tgfr lizr tgfr chicken TGFBR mouse zer finch humn Tgfr TGFBR TGFBR Tgfrs mek tgfr stickleck fugu mek humn zerfish Enoglins opossum zer finch mouse Eng Xenopus / um DMSO BCA PCS PCS PHBC BA PHBC DMSO CtAs DMSO DMSO e / PCS PHBC CtAs um Nifeipine BCA PCS BA PHBC Nifeipine Nifeipine Nifeipine Supplementry Figure Ientifiction of zerfish enoglin orthologue. () Schemtic representtion of the enoglin locus showing conservtion of gene orer (synteny) etween humn chromosome q- n two prlogous loci on zerfish chromosome contining the Pregnncy-ssocite plsm protein A (ppp), Astrotctin (stn), retrotrnspose Atril myosin light chin (myl-rt), Delete in ler cncer (c), enoglin (), enylte kinse (k) n G-protein signling moultor (gpsm) genes. Prlogous genes tht were likely uplicte uring the teleost whole genome upliction (WGD) event re inicte in lue. Susequent gene loss on the prlogous chromosomes (lost genes in grey) most likely resulte in the current gene orer. Pink shing inictes smll synteny lock contining the Enoglin n AK genes. Dshe lines inicte synteny reks etween humn n zerfish. () Alignment of the conserve C-terminl region of zerfish n humn Enoglin. Asterisks inicte conserve resiues. (c) Phylogenetic tree se on the lignment of the C-terminl sequences (trnsmemrne region n C-terminl omin) of verterte Tgfr (green shing) n Enoglin (re shing). Numers t rnchpoints represent ootstrp vlues (percentge out of replictes). () Whole-mount in situ hyriiztion of shows vsculrrestricte expression in hpf zerfish emryos. Expression is etecte in the sl communicting rtery (BCA), the posterior communicting segments (PCS), silr rtery (BA), primoril hinrin chnnels (PHBC) n centrl rteries (CtAs) of the hinrin s well s the orsl ort (, rrows), posterior crinl vein () n orsl longituinl nstomotic vessel (DLAV, rrowhes) of the trunk vsculture. Scle r in overview picture is um, in high mgnifiction imges scle r is um. Imges re representtive of out of emryos. (e) Whole-mount in situ hyriiztion of expression in WT emryos trete with um nifeipine from - hpf. Note tht loo flow reuction oes not ffect hinrin expression, ut ecreses mrna levels in the DLAV (rrowhes). Scle r in overview picture is um, in high mgnifiction imges scle r is um. Imges re representtive of out of emryos. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

2 qpcr * rteril ECs venous ECs Fol chnge WT mu hpf WT mrna mu mu +/+ +/+ / / +/- +/- / / -/- -/- / / Supplementry Figure mrna is suject to nonsense-meite ecy in mu mutnts. () qpcr to etect zerfish in FACSsorte rteril n venous ECs from poole hpf emryos. Dt is shown s reltive expression normlize to WT rteril EC levels. In WTs, trnscript cn e etecte t similr levels in the rteril n venous popultions, n is lmost non-etectle in mu mutnt ECs. Experiments were performe n= times inepenently n were nlyse y pire Stuent s t-test. () Whole-mount in situ hyriiztion for enoglin expression t hpf in emryos from WT, mu n mu heterozygous incrosses. All emryos crrying mu lleles (which contin nt sustitution tht oes not introuce frmeshift) show the sme stining intensity s WT, with prticulrly strong expression in the (rrowhe). mu +/- emryos lrey show ecrese stining, which isppers lmost entirely from the he n trunk vsculture in mu mutnts (rrows). Scle r is um. Imges re representtive of the numer of emryos s inicte in the respective pnel. n.s., not significnt, *P<., **P<., P<., error rs inicte s.e.m. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

(, ) Mximum intensity projections of confocl z-stcks of pf WT () n mu mutnts ().

(c-f) Quntifiction of ISV ientity (c), visv ientity (), ISVs contining DsRe+ erythrocytes (e) n remoelle ISVs (f).

3 wiltype mu Tg(krl:EGFP) s merge pf pf Tg(gt:DsRe) s c e n.s. f % of totl ISVs ISVs visvs ISVs with RBCs Remoelle ISVs Arteril ISVs Venous ISVs n.s. n.s. sis mu sis mu sis mu sis mu % of totl ISVs % of totl ISVs % of totl ISVs Supplementry Figure ISV remoelling in WT n mu pf juveniles. (, ) Mximum intensity projections of confocl z-stcks of pf WT () n mu mutnts (). Yellow rrowhes point to remoelle ISVs tht no longer mintin connection to the DLAV. Scle r is um. (c-f) Quntifiction of ISV ientity (c), visv ientity (), ISVs contining DsRe+ erythrocytes (e) n remoelle ISVs (f). WTs n mu mutnts hve no ifference in ISV istriution or presence of RBCs, ut mu mutnts show increse remoelling (n= silings ( ISVs nlyse) n n= muts ( ISVs nlyse)). Anlyse y unpire Stuent s t-test. n.s., not significnt, *P<., **P<., P<., error rs inicte s.e.m. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

WT % ISVs/segment n= ISVs mu % ISVs/segment n= ISVs % visvs/segment n= ISVs % visvs/segment n= ISVs c Tg(krl:Hs.

) f mu % ISVs with RBC flow/segment n= ISVs mu + Tg(fli:-GFP) mu rescue construct Dimeter (um) mu mu + Tg(fli:-GFP) mu Supplementry Figure Arteril-venous specifiction is unffecte in enoglin mutnts.

WTs n mutnts hve similr istriution of rteries n veins, with slight increse in the numer of unresolve ISV connections in mu mutnts. Inicte numers of ISVs from WT n mut emryos were nlyse.

4 WT % ISVs/segment n= ISVs mu % ISVs/segment n= ISVs % visvs/segment n= ISVs % visvs/segment n= ISVs c Tg(krl:Hs.HRAS-mCherry) s ; Tg(fli:-GFP) mu % unresolve ISVs/segment mu n= ISVs GFP negtive GFP positive % unresolve ISVs/segment Dimeter (um) e n= ISVs imeter mu mu + Tg(fli:-GFP) mu imeter n.s. (p=.) f mu % ISVs with RBC flow/segment n= ISVs mu + Tg(fli:-GFP) mu rescue construct Dimeter (um) mu mu + Tg(fli:-GFP) mu Supplementry Figure Arteril-venous specifiction is unffecte in enoglin mutnts. (, ) Arteril-venous ientity of ISVs s etermine y flow irection n/or morphology (connection to or ) in WTs n mu mutnts. WTs n mutnts hve similr istriution of rteries n veins, with slight increse in the numer of unresolve ISV connections in mu mutnts. Inicte numers of ISVs from WT n mut emryos were nlyse. (c) Mximum intensity projection of confocl z-stck of mu mutnts t hpf. Tg(fli:-GFP) mu emryos exhiit vsculr-specific GFP expression, evience y overlp with Tg(krl:Hs. HRAS-mCherry) s. Scle r is um. (, e) Quntifiction of () n (e) imeters in mu mutnts with or without the Tg(fli:- GFP) mu rescue construct. Tgge enoglin-gfp moestly rescues, ut not imeters (n= GFP-negtive, n= GFP-positive mu mutnts). Anlyse y unpire Stuent s t-test. (f) Quntifiction of the numer of ISVs ctively crrying RBCs shows rescue in mu emryos crrying the Tg(fli:-GFP) mu rescue construct. ISVs from GFP-positive mu mut emryos were nlyse. n.s., not significnt, *P<., **P<., P<., error rs inicte s.e.m. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

ATG klf promoter Tg(krl:Hs.HRAS-mCherry) s ; Tg(klf:YFP) mu Tg(klf:YFP) mu hpf citrine pa knr CH-A ~ k ng ctr MO c ng tnnt MO Fluorescence intensity (.u.) intensity * e Fluorescence intensity (.u.) intensity ** f Fluorescence intensity (.

A Citrine cssette ws inserte ehin the enogenous ATG of the klf gene contine in the CH-A BAC y homologous recomintion.

5 ATG klf promoter Tg(krl:Hs.HRAS-mCherry) s ; Tg(klf:YFP) mu Tg(klf:YFP) mu hpf citrine pa knr CH-A ~ k ng ctr MO c ng tnnt MO Fluorescence intensity (.u.) intensity * e Fluorescence intensity (.u.) intensity ** f Fluorescence intensity (.u.) ISV intensity n.s. (p=.) ctr MO tnnt MO ctr MO tnnt MO ctr MO tnnt MO Supplementry Figure Genertion of the Tg(klf:YFP) mu reporter line. () Schemtic of the cloning strtegy. A Citrine cssette ws inserte ehin the enogenous ATG of the klf gene contine in the CH-A BAC y homologous recomintion. (, c) Tg(klf:YFP) mu emryos isply vsculr-specific YFP expression, evience y overlp with Tg(krl:Hs. HRAS-mCherry) s. Emryos trete with tnnt MO lose YFP expression in the n. Scle r is um. (-f) Quntifiction of YFP fluorescence intensity in the, n ISVs in ctr MO n tnnt MO injecte emryos (/: n= control MO, n= tnnt MO; ISVs: n= ISVs from control MO, n= ISVs from tnnt MO). Anlyse y Mnn-Whitney U test. n.s., not significnt, *P<., **P<., P<., error rs inicte s.e.m. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

S U P P L E M E N TA R Y I N F O R M AT I O N Mosic

Dimeter (um) WT --> Mut c f Host Donor ** mu EC WT EC

WT -> Mut g WT lumen Host Host Donor Mosic (), while

mosic ISVs from () WT->WT, () Mut->WT n (c) WT->Mut

Opticl sections revel portions of lumen comprise only

cellulr composition from () WT->WT, (e) Mut->WT n (f)

imeter when they forme ll of the lumen in WT->Mut

(WT>WT n= vessel segments from ISVs; Mut->WT n= vessel

6 S U P P L E M E N TA R Y I N F O R M AT I O N Mosic vessels Host Overly WT --> WT Donor Lumen x-section WT -> WT n.s. (p=.) Dimeter (um) * Mut --> WT e Donor Mosic Mut -> WT n.s. (p=.) Dimeter (um) WT --> Mut c f Host Donor ** mu EC WT EC mu lumen Mosic lumen Dimeter (um) ISV lumen size Mosic WT -> Mut g WT lumen Host Host Donor Mosic (), while mutnt cells cuse iltion Sugen etnorml l.,imeters Supplementry Fig.whenthey forme prt or ll of the lumen in Mut->WT situtions (e). WT cells were le to reuce Supplementry Figure Trnsplnttion nlysis of mosic ISVs. (-c) Mximum intensity projections of confocl z-stck of mosic ISVs from () WT->WT, () Mut->WT n (c) WT->Mut trnsplnttions. Opticl sections revel portions of lumen comprise only of onor cells, only of host cells, or mosic comintion of host n onor cells. Scle r on overview is um, scle r on lumen cross-section is um. (-f) Quntifiction of mosic ISV imeters epening on cellulr composition from () WT->WT, (e) Mut->WT n (f) WT->Mut trnsplnttions. All ISVs from WT->WT trnsplnttions h ISV imeter when they forme ll of the lumen in WT->Mut scenrios (f). (WT>WT n= vessel segments from ISVs; Mut->WT n= vessel segments from ISVs; WT->Mut n= vessel segments from ISVs). Anlyse y One-Wy ANOVA. (g) Schemtic summrizing the requirement for function in every cell of vsculr tue to mintin proper imensions. n.s., not significnt, *P<., **P<., P<., error rs inicte s.e.m. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

7 Moel c hpf cpillry flow wek hpf cpillry flow strong WT No hpf Growth n flow-inuce outwr remoelling flow-inuce EC cell shpe chnges EC res constnt F pressure No increse in EC re sher stress F cpillry flow wek EC res increse impire cell shpe chnges -/- Sugen et l., Supplementry Fig. Supplementry Figure Moel for enoglin s role in the iphsic response of ECs to loo flow. () By hpf, the first rtery n vein hve ssemle y vsculogenesis. Bloo flow strts. () As evelopment procees, cric output rises. The xil vessels increse in imeter n crry n incresing volume of loo. At hpf, the cpillries connecting the rtery n vein hve just egun to lumenize n crry flow. (c) Between n hpf, loo flow triggers shpe chnges in WT ECs. They elongte n lign in the irection of flow, without chnging their surfce res. This lthens the vessel n reuces its imeter. The rteril constriction reirects loo flow through the cpillry network. () In -eficient loo vessels, ECs re unle to limit their surfce res in response to loo flow. Although elongtion still occurs to some extent, the increse in size impees the ility of ECs to lign to the flow. Ultimtely, this results in vessel iltion n the formtion of n rterio-venous shunt. The high velocities chieve in the shunt prevent RBCs from pssing into cpillries, n the pulstile qulity of rteril flow is trnsmitte to the venous return. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

8 Originl lot scns use in Figure e α-tuulin Neg-si -si -si Supplementry Figure Originl scnne lots use in Figure e. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

9 Supplementry Movie Legens Supplementry Movie Distl loo flow in WT fin regenerte t p. Brightfiel movie of loo flow in the istl regenerting fin of WT fish t p. Numers lel iniviul ony rys. Arrows inicte flow irection, while she lines enote rteries (re) n veins (lue). from centrlly locte rtery istriutes through plexus to two veins t the en of ech ry. Note wek flow reversl in lterl vein of the first ry, rcing over n inctive rtery. Supplementry Movie AVMs n isture flow in mu fin regenerte t p. Brightfiel movie of loo flow in the istl regenerting fin of mu mutnt t p. Numers lel iniviul ony rys. Arrows inicte flow irection, while she lines enote rteries (re) n veins (lue). Note prominent AVMs in secon ry from the left. In ry #, more proximlly locte AVMs either stop or reverse loo flow in the veins, n the centrl rtery is not ctive. Ry # hs strong flow reversl in the left vein, rcing over n inctive centrl rtery. Note leeings t istl en of ll rys. Supplementry Movie ISV flow in hpf siling emryo. Brightfiel movie of RBC flow in the trunk of siling emryo t hpf. Nerly ll ISVs on oth sies of the emryo crry RBC flow. Supplementry Movie ISV flow in hpf mu emryo. Brightfiel movie of RBC flow in the trunk of mu emryo t hpf. Note tht only or ISVs hve persistent RBC flow, while most hve no or only very intermittent RBC flow. Supplementry Movie Opticl ril restores RBC flow in cpillries of mu mutnts. Brightfiel movie of RBC flow in the trunk of mu emryo t hpf. Hologrphic opticl tweezers (HOT) re focuse t entrnce to n ISV, ut lser is turne off. Aout hlfwy through movie the lser is turne on (re circle inictes HOT focl point), slows own RBCs tht pss through the lser, n iverts them from the min rteril loo flow into the cpillry. Supplementry Movie Time-lpse of cell shpe chnges in etween - hpf in siling emryo. Mximum intensity projection of confocl z-stcks of Tg(fli:lifectEGFP) mu ; Tg(fli:nEGFP) y, n Tg(-.flt:RFP) hu in siling emryo etween - hpf. Acquisitions were me t min intervls, shown t frmes per secon. Note ecrese in imeter etween - hpf, n elongtion of ECs. Supplementry Movie Time-lpse of cell shpe chnges in etween - hpf in mu emryo. Mximum intensity projection of confocl z-stcks of Tg(fli:lifectEGFP) mu ; Tg(fli:nEGFP) y, n Tg(-.flt:RFP) hu in mu emryo etween - hpf. Acquisitions were me t min intervls, shown t frmes per secon. Note similr imeter t hpf s siling in Supplementry Movie tht fils to ecrese pprecily y hpf. Note, itionlly, the rmtic increse in EC sizes compre to silings uring the movie. Mcmilln Pulishers Limite, prt of Springer Nture. All rights reserve.

Supporting Information. Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-like

Supporting Information. Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-like Supporting Informtion Cytosolic Irrdition of Femtosecond Lser Induces Mitochondri-dependent Apoptosis-like Cell Deth vi Intrinsic Rective Oxygen Cscdes Jonghee Yoon 1,2, Seung-wook Ryu 1,3, Seunghee Lee