Capsaspora Owczarzaki

Home , Dermocystida, Filasterea

The origin of metazoan multicellularity: what comparative genomics is telling us and how we could go forward Iñaki Ruiz-Trillo, Institute of Evolutionary Biology & Universitat de Barcelona & Kitp

Cooperation And The Evolution Of Multicellularity, Kitp, 21 February 2013

1 Institut de Biologia Evolutiva (UPF-CSIC)

2 3 3 3 3 last common unicellular ancestor

4 last common unicellular ancestor

4 1. A phylogenetic framework

5 who are the closest living relatives of animals?

Fungi Choanoﬂagellata Metazoa

6 who are the closest living relatives of animals?

Fungi Choanoﬂagellata Metazoa

Capsaspora

Ministeria

Nuclearia Ichthyosporea ? Corallochytrium

6 A new phylogenetic framework

Fungi Choanoﬂagellata Metazoa

Holozoa

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2012 Mol Biol Evol 29, 531–544.

7 A new phylogenetic framework

Fungi Choanoﬂagellata Metazoa Nucleariidae

Fonticula alba

Holozoa

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2012 Mol Biol Evol 29, 531–544.

7 A new phylogenetic framework

Fungi Choanoﬂagellata Metazoa Nucleariidae

Filasterea Capsaspora Ministeria

Fonticula alba

Holozoa

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2012 Mol Biol Evol 29, 531–544.

7 A new phylogenetic framework

Fungi Choanoﬂagellata Metazoa Nucleariidae

Filasterea Capsaspora Ministeria

Fonticula alba

Ichthyosporea

Holozoa

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2012 Mol Biol Evol 29, 531–544.

7 2. Genome data

8 M. brevicollis genome

Fungi Choanoﬂagellata Metazoa

King et al. 2008 Nature 451:783-788.

9 M. brevicollis genome

Fungi Choanoﬂagellata Metazoa

King et al. 2008 Nature 451:783-788.

9 M. brevicollis genome

Fungi Choanoﬂagellata Metazoa

cell adhesion (cadherins) cell signaling (protein tyrosine kinases)

King et al. 2008 Nature 451:783-788.

9 M. brevicollis genome

Fungi Choanoﬂagellata Metazoa

cell adhesion (cadherins) cell signaling (protein tyrosine kinases) integrins WNT pathway TGF-B pathway Notch pathway Hedgehog pathway several TFs

King et al. 2008 Nature 451:783-788.

9 Unicellular relatives of animals: not a game of one

Fungi Holozoa Choanoﬂagellata Metazoa Nucleariidae

Filasterea Capsaspora Ministeria

Fonticula alba

Ichthyosporea

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2011 Mol Biol Evol

10 Unicellular relatives of animals: not a game of one

Fungi Holozoa Choanoﬂagellata Metazoa Nucleariidae

Filasterea Capsaspora Ministeria

Fonticula alba

Ichthyosporea

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2011 Mol Biol Evol

10 Unicellular relatives of animals: not a game of one

Fungi Holozoa Choanoﬂagellata Metazoa Nucleariidae

Filasterea Capsaspora Ministeria

Fonticula alba

Ichthyosporea

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2011 Mol Biol Evol

10 Unicellular relatives of animals: not a game of one

Fungi Holozoa Choanoﬂagellata Metazoa Nucleariidae

Filasterea Capsaspora Ministeria

Fonticula alba

Ichthyosporea

Ruiz-Trillo et al. 2008 Mol Biol Evol 25:664-672. Shalchian-Tabrizi et al. 2008 PLoS ONE 3:e2098. Brown et al. 2009 Mol Biol Evol 26:2699-2709. Torruella et al. 2011 Mol Biol Evol

10 UNICORN project

Fungi Holozoa Allomyces macrogynus Choanoﬂagellata Salpingoeca Monosiga Spizellomyces Filasterea rosetta brevicollis Metazoa punctatus Capsaspora Ministeria Mortierella owczarzaki vibrans verticillata

Ichthyosporea Nucleariidae Sphaeroforma Amoebidium arctica parasiticum Fonticula alba

Thecamonas Apusozoa trahens Ruiz-Trillo et al. 2007. Trends in Genetics 23:113-118. 11 UNICORN: sequenced genomes (August 2012)

Fungi Allomyces macrogynus Choanoﬂagellata Monosiga Spizellomyces Salpingoeca Filasterea brevicollis Metazoa punctatus rosetta Capsaspora Ministeria Mortierella owczarzaki vibrans verticillata

Ichthyosporea Nucleariidae Sphaeroforma Amoebidium arctica parasiticum Fonticula alba

we are responsible for Capsaspora Thecamonas Apusozoa and trahens Sphaeroforma

Ruiz-Trillo et al. 2007. Trends in Genetics 23:113-118. 12 Integrin-mediated adhesion system: pre-UNICORN view

Amoebozoa Thecamonas Fungi Capsaspora Choanoﬂagellata Metazoa Salpingoeca Allomyces Monosiga

Spizellomyces Batrachochytrium

membrane

13 Integrin-mediated adhesion system: post-UNICORN view

Amoebozoa Thecamonas Fungi Capsaspora Choanoﬂagellata Metazoa Allomyces Monosiga Salpingoeca

Spizellomyces Batrachochytrium

membrane

Sebé-Pedrós et al. 2010. PNAS 107:10142-10147.

14 Metazoan Transcription Factors (TFs)

Other Other eukaryotes Amoebozoa Amastigomonas fungi Allomyces Spizellomyces Capsaspora Monosiga Metazoa HD: PBC, prd-like. Ets, Smad, NR HD: ANTP, POU, LIM, Irx, Meis. bZIP: PAR, C/EBP, CREB. bZIP: Jun, Fos, Atf3, ... Grainyhead-like bHLH: Groups A, D, E, F. bHLH: MAD, MITF, Class I Fox TFs USF, Mlx, Group C. HMGbox: Sox, TCF/LEF.

bZIP: Atf4/5 HMGbox: Sox-like Holozoa

Grainyhead-like Runx NFkappaB/Rel p53

bZIP: Oasis, Atf6, CREB, PAR, C/EBP, Atf2. Opisthokonts bHLH: Myc, Max, Mad, USF, Mlx, SREBP, MITF, Group C: ARNT. HD: Pbx, Prd-like. T-box Unikonts Metazoan-like Mef2 CP2 TFs

Metazoan-like STAT

Fox TFs STAT TFs Churchill

Mef2-like (Type II) MAD-box bZIP TFs bHLH TFs HD: TALE, non-TALE HMGbox TFs

Sebé-Pedrós et al. 2011. Mol. Biol. Evol. 28:1241-54 15 Metazoan Transcription Factors: pre-UNICORN view

Metazoa

Capsaspora Choanoﬂagellata NF-Kappa Thecamonas Monosiga Allomyces Fungi ETS, Smad, NR Grainyhead-like T-box Spizellomyces Amoebozoa Runx

p53

other eukaryotes

Fox

membrane

16 Metazoan Transcription Factors: post-UNICORN view

Metazoa

Capsaspora Choanoﬂagellata Thecamonas Monosiga Allomyces Fungi ETS, Smad, NR

Spizellomyces Amoebozoa

other Grainyhead-like eukaryotes p53 Runx

NF-Kappa T-box

membrane

Fox

Sebé-Pedrós et al. 2011. Mol. Biol. Evol. 28:1241-54 17 Capsaspora has a Brachyury gen

Brachyury Fungi Co Dtbx1 Dm Bra

Filasterea Sd Bra Porifera Hs Tbx19 Om Bra Cnidaria & Ctenophora

Pp Bra Nv Bra Co Bra Placozoa Bra Ml Ta Bra Hv Bra2 Bilateria Co Dtbx2 Hs T Pc Bra Sr Bra Hv Bra1He Bra Aq Tbx1/15/20 Av Tbx1/15/20 Sp Tbx

100/1.00

60/0.98

50/0.98 Tbx2/3 64/1.00 Tbr Dm bi 34/0.84 Hs Tbx21HsEomes Pp Tbx2/3 Ta Tbx2/3 Hs Tbx3 100/1.00 Hs Tbr1 ! Nv Tbx2/3 Hs Tbx2d Ml Tbx2/3

77/1.00 -/0.90 -/- -/1.00 Aq TbxA Aq TbxE Hs Tbx15 Nv Tbx20 67/0.99 85/1.00 Aq TbxC Hs Tbx18 41/1.00 Aq TbxD

Hs Tbx22 Nv Tbx20.3 Hs Tbx6 Hs Tbx5 Hs Tbx4 Hs MGA Nv Tbx20.2 Hs Tbx20

Pc Tbx4/5 Dm dc2

Aq Tbx4/5 Dm dc1 Ml Tbx1 Dm dc3 Dm midline

Nv Tbx1p Tbx2 Sd Tbx6 Aq TbxB Dm H15 Hs Tbx1 Nv Tbx4/5 Nv Tbx1 Nv Tbx20.1

Dm org-1

Tbx1/15/20 Hs Tbx10 Tbx4/5

Co Tbx3

0.4

Sebé-Pedrós et al. 2011. Mol. Biol. Evol. 28:1241-54 18 PFAM gain/loss

a i k b [Gain] Gene Ontology N domains za ollis r c Holozoa Signal transducer activity 8 oa i z evi cza Transcription regulatory region DNA binding 7 r w g eta o un Metazoa Extracellular region 9 . F M M. b C Outgroup Transcription regulatory region DNA binding 8

c [Loss] 5157 3014 3287 3941 +235 +19 +6 +55 Holozoa Hydrolase activity (O-glycosyl bonds) 7 -56 -1983 -1688 -1143 Fungi Multi-organism process 10 Negative regulation of hydrolase activity 7 Transcription regulatory region DNA binding 16 Metazoa+ 4978 Choano +61 M. brevicollis Extracellular region 29 -52 Regulation of transcription, DNA-dependent 62 Nucleoplasm 22 Holozoa 4969 Developmental process 11 +100 Cellular cell wall organization or biogenesis 7 -160 Racemase and epimerase activity 8 C. owczarzaki Extracellular region 26 5029 Opisthokonta Receptor binding 12 Reproduction 12 Multicellular organismal process 13 Cell wall organization biogenesis 11 Pathogenesis 8 Viral reproduction 9 External encapsulating structure 7

Figure 2. Suga et al. PFAM gain/loss

a i k b [Gain] Gene Ontology N domains za ollis r c Gains (geneHolozoa ontology,Signal transdu >cer 7 a domainsctivity ) 8 oa i z evi cza Transcription regulatory region DNA binding 7 r w g eta o un HolozoaMetazoa Extracellular region 9 . F M M. b C Outgroup Transcription regulatory region DNA binding 8 Signal transducer activity c [Loss]Transcription regulatory 5157 3014 3287 3941 +235 +19 +6 +55 regionHolozoa DNAHydrolase binding activity (O-glycosyl bonds) 7 -56 -1983 -1688 -1143 Fungi Multi-organism process 10 Negative regulation of hydrolase activity 7 Transcription regulatory region DNA binding 16 Metazoa+ 4978 Choano +61 M. brevicollis Extracellular region 29 -52 Regulation of transcription, DNA-dependent 62 Nucleoplasm 22 Holozoa 4969 Developmental process 11 +100 Cellular cell wall organization or biogenesis 7 -160 Racemase and epimerase activity 8 C. owczarzaki Extracellular region 26 5029 Opisthokonta Receptor binding 12 Reproduction 12 Multicellular organismal process 13 Cell wall organization biogenesis 11 Pathogenesis 8 Viral reproduction 9 External encapsulating structure 7

Figure 2. Suga et al. PFAM gain/loss

a i k b [Gain] Gene Ontology N domains za ollis r c Gains (geneHolozoa ontology,Signal transdu >cer 7 a domainsctivity ) 8 oa i z evi cza Transcription regulatory region DNA binding 7 r w g eta o un HolozoaMetazoa Extracellular region 9 . F M M. b C Outgroup Transcription regulatory region DNA binding 8 Signal transducer activity c [Loss]Transcription regulatory 5157 3014 3287 3941 +235 +19 +6 +55 regionHolozoa DNAHydrolase binding activity (O-glycosyl bonds) 7 -56 -1983 -1688 -1143 Fungi Multi-organism process 10 Metazoa Negative regulation of hydrolase activity 7 Transcription regulatory region DNA binding 16 Metazoa+ 4978 Choano +61 M.Extracellular brevicollis Extracellular region region 29 -52 Regulation of transcription, DNA-dependent 62 TranscriptionNucleoplasm regulatory 22 Holozoa 4969 region DNADevelopmental binding process 11 +100 Cellular cell wall organization or biogenesis 7 -160 Racemase and epimerase activity 8 C. owczarzaki Extracellular region 26 5029 Opisthokonta Receptor binding 12 Reproduction 12 Multicellular organismal process 13 Cell wall organization biogenesis 11 Pathogenesis 8 Viral reproduction 9 External encapsulating structure 7

Figure 2. Suga et al. PFAM gain/loss

a i k b [Gain] Gene Ontology N domains za ollis r c Gains (geneHolozoa ontology,Signal transdu >cer 7 a domainsctivity ) 8 oa i z evi cza Transcription regulatory region DNA binding 7 r w g eta o un HolozoaMetazoa Extracellular region 9 . F M M. b C Outgroup Transcription regulatory region DNA binding 8 Signal transducer activity c [Loss]Transcription regulatory 5157 3014 3287 3941 +235 +19 +6 +55 regionHolozoa DNAHydrolase binding activity (O-glycosyl bonds) 7 -56 -1983 -1688 -1143 Fungi Multi-organism process 10 Metazoa Negative regulation of hydrolase activity 7 Transcription regulatory region DNA binding 16 Metazoa+ 4978 Choano +61 M.Extracellular brevicollis Extracellular region region 29 -52 Regulation of transcription, DNA-dependent 62 TranscriptionNucleoplasm regulatory 22 Holozoa 4969 region DNADevelopmental binding process 11 +100 Cellular cell wall organization or biogenesis 7 -160 Racemase and epimerase activity 8 C. owczarzaki Extracellular region 26 5029 Loss Opisthokonta Receptor binding 12 Reproduction 12 M. brevicollisMulticellular organismal process 13 Cell wall organization biogenesis 11 RegulationPathogenesis of transcription, 8 DNA-dependentViral reproduction 9 External encapsulating structure 7

Figure 2. Suga et al. assa wczarzaki r ectensis alciparum o v f adhaerens A. thaliana A. queenslandica E. siliculosus P. tetraurelia L. major P. C. reinhardtii E. histolytica D. discoideum L. biclor N. c C. M. brevicollis T. N. H. magnipapillata C. elegans D. melanogaster H. sapiens )LErLQRJHQBDEJB& Coagulation_fDFBí&BW\SHBGRP Tetraspanin_EC2 7KURPERVSRQGLQBBrpt Collagen Laminin_G Zona_pellucida_Endoglin/CD105 VWF_C Cell adhesion TIL_dom KrLQJOHíOLke and extracellular components Pentaxin EGF_laminin Marvel CadherLQíOLke ,JíOLke_fold )LEURQHFWLQBW\SH &DO[BEHWD (*)íOLke &íW\SHBOHFWLQíOLke LDrepeatLR_classA_rpt Sushi_SCR_CCP EGF_rcpt_L LDLR_classB_rpt Extracellular region of receptors NHL_repeat and ligands GPS_dom 6UFUBUFSWíUHO CUB *DODFWRVHíEGíOLke &\Vírich_flank_reg_C &KLWLQíEGBGRP MAM_dom Wnt 7*)íEB& P$1íBGRPDLQ GPCR_Rhodpsn_supfam *3&5BBVHFUHWLQíOLke Other receptors of GPCR_3 7TM_GPCR_serpentine_rcpt_Srw extracellular signals Nucl_hrPQBUFSWBOLJíEGBFRUH Neur_channel Na+channel_ASC K_chnl Channels and transporters Na/ntran_symport OA_transporter gene expansions Tyr_kinase_cat_dom Tyr_PDVHBUFSWQRQíUFSW PH_like_dom SH2 SH3_domain PTyr_interaction_dom Signal transduction in cell PDZ 75$)íOLke &+.BNLQDVHíOLke Znf_C2H2 Znf_C2CH =QIB&+&B5,1*íW\SH Znf_NHR/GATA Znf_TRAF Zinc fingers assa wczarzaki r ectensis Znf_TTF alciparum o v f Znf_AD adhaerens =QIB%íEox A. thaliana A. queenslandica

E. siliculosus P. tetraurelia L. major P. C. reinhardtii E. histolytica D. discoideum L. biclor N. c C. M. brevicollis T. N. H. magnipapillata C. elegans D. melanogaster H. sapiens Znf_BED_prd Homeodomain )LErLQRJHQBDEJB& TF_fork_head Coagulation_fDFBí&BW\SHBGRP SíOLkHB7)B'1$íEG Tetraspanin_EC2 7)B7íEox 0$')íGRP Transcriptional control 7KURPERVSRQGLQBBrpt Tscrpt_reg_SCAN Collagen HTH_Psq Ets Laminin_G %Eox_C Zona_pellucida_Endoglin/CD105 Band_41_domain VWF_C )LODPLQ$%3BUHSHDWíOLke Cytoskelton and its control Cell adhesionF (Filament) TIL_dom Spectrin_repeat KrLQJOHíOLke and extracellularCARD components Pentaxin DEA7+íOLke Pept_C14_p45 EGF_laminin Pept_cys/ser_Tr\SVLQíOLke Marvel Peptidase_M12A Protein degradation/modification CadherLQíOLke Peptidase_M12B and apoptosis Peptidase_M13_N ,JíOLke_fold Prot_inh_KXQ]íP )LEURQHFWLQBW\SH .D]DOíW\SHBGRP &DO[BEHWD ShK_toxin IntegrDVHBFDWíFRUH (*)íOLke DDE_SF_endonXFOHDVHB&(13%íOLke &íW\SHBOHFWLQíOLke DNA_helicase_PIF1 Functions on DNA or Alkaline_PDVHíOLkHBDED RNA molecules LDrepeatLR_classA_rpt Histone_core_D Sushi_SCR_CCP 51DVH+íOLke_dom EGF_rcpt_L Retrotrans_Pao ExtracellularTrDQVSRVDVHB7FíOL region kofe receptorsVirus and transposons LDLR_classB_rpt HATC NHL_repeat and ligandsRVT GPS_dom /HXírich_rSWBW\SLFDOíVXEW\S /55íFRQWDLQB1 6UFUBUFSWíUHO BTB/POZ_fold CUB BACK *DODFWRVHíEGíOLke KHOFKíOLke_gigaxonin Ankyrin_rpt Other/diverse function &\Vírich_flank_reg_C SOCS_C &KLWLQíEGBGRP $OSKDíFrystallin/HSP MAM_dom Butyrophylin NACHT_NTPase Wnt DUF1759

7*)íEB& a al r f ec wc v

PEsil $1íBGRPDLQ adh P o Ptet Crei /ELF Ehis Ddis Atha Cele Nc T Lmaj N 0EUH Dmel Aque Hsap GPCR_Rhodpsn_supfam C Hmag *3&5BBVHFUHWLQíOLke Other receptors of GPCR_3 0 1 Normalized gene count 7TM_GPCR_serpentine_rcpt_Srw extracellular signals Nucl_hrPQBUFSWBOLJíEGBFRUH 20 Neur_channel Na+channel_ASC K_chnl Channels and transporters Na/ntran_symport OA_transporter Tyr_kinase_cat_dom Tyr_PDVHBUFSWQRQíUFSW Figure 3 Suga et al. PH_like_dom SH2 SH3_domain PTyr_interaction_dom Signal transduction in cell PDZ 75$)íOLke &+.BNLQDVHíOLke Znf_C2H2 Znf_C2CH =QIB&+&B5,1*íW\SH Znf_NHR/GATA Znf_TRAF Zinc fingers Znf_TTF Znf_AD =QIB%íEox Znf_BED_prd Homeodomain TF_fork_head SíOLkHB7)B'1$íEG 7)B7íEox 0$')íGRP Transcriptional control Tscrpt_reg_SCAN HTH_Psq Ets %Eox_C Band_41_domain )LODPLQ$%3BUHSHDWíOLke Cytoskelton and its control F (Filament) Spectrin_repeat CARD DEA7+íOLke Pept_C14_p45 Pept_cys/ser_Tr\SVLQíOLke Peptidase_M12A Protein degradation/modification Peptidase_M12B and apoptosis Peptidase_M13_N Prot_inh_KXQ]íP .D]DOíW\SHBGRP ShK_toxin IntegrDVHBFDWíFRUH DDE_SF_endonXFOHDVHB&(13%íOLke DNA_helicase_PIF1 Functions on DNA or Alkaline_PDVHíOLkHBDED RNA molecules Histone_core_D 51DVH+íOLke_dom Retrotrans_Pao TrDQVSRVDVHB7FíOLke Virus and transposons HATC RVT /HXírich_rSWBW\SLFDOíVXEW\S /55íFRQWDLQB1 BTB/POZ_fold BACK KHOFKíOLke_gigaxonin Ankyrin_rpt Other/diverse function SOCS_C $OSKDíFrystallin/HSP Butyrophylin NACHT_NTPase DUF1759 a al r f ec wc v Esil adh P o Ptet Crei /ELF Ehis Ddis Atha Cele Nc T Lmaj N 0EUH Dmel Aque Hsap C Hmag 0 1 Normalized gene count

Figure 3 Suga et al. Capsaspora cell

Cell adhesion Extracellular Matrix Cadherin Integrins DG SSPN SG IgCAM C-Lectin Catenins Src FAK DP DB SYN ILK/PINCH/Parvin Ras Talin/Vinculin/Paxillin Smoothened MAPK MAPKK MAPKKK RTKs Hedge Hog Patched Hedge Hog Nuclear Receptor JNK CTKs Dispatched Gli pTyr signaling JAK p53 Runt 1)ț% Grh STAT NRPTPs RPTP Notch Myc Brachyury Fringe Notch CSL Delta Jun Fos Sox Ets SMAD TGFβ 7*)ȕ5 7*)ȕ HD (TALE) HD (non-TALE) Noggin Rhodopsin 7TM S6K p70 TOR GluR Lef-1/Tcf Sd/TEAD AKT 4E-BP1 Raptor Secretin *Į *ȕ *Ȗ Yorkie/YAP ITR RGS Hippo/Mst Merlin GPR108 PDE Growth Sav Kibra FAT OA1 Regulation Warts/Lats Expanded Dsh Axin Wnt Frizzled ȕ&DWHQLQ Mats/Mob1 CK1 GSK3

21 Protein Tyrosine Kinases-Metazoan PTKs

Conserved among all metazoans!

Choanoﬂagellates have hundreds of PTKs

22 PTKs-Cytosolic PTKs

Metazoa Capsaspora

H. Suga et al., Science Signaling 5, ra35 (2012). 23 PTKs-Receptor PTKs

Metazoa Capsaspora

No homolog!

H. Suga et al., Science Signaling 5, ra35 (2012). 24 PTKs-lineage-specific divergence of RPTKs

M. brevicollis A. queenslandica

M. brevicollis

A. queenslandica 11 19 1 11 4 6 C. owczarzaki 4 1 2 6 1 1 5 1 4 human 1 3 19 D. melanogaster human D. melanogaster C. owczarzaki

Cytoplasmic Receptor

Fig. 5. Suga et al.

H. Suga et al., Science Signaling 5, ra35 (2012). 25 3. Functional analyses-model organism

26 Filasterea: Capsaspora owczarzaki

27 Capsaspora owczarzaki attached/filopodial stage

28 Capsaspora owczarzaki attached/filopodial stage

28 Capsaspora owczarzaki cystic/floating stage

29 Capsaspora owczarzaki aggregative stage

30 Capsaspora owczarzaki aggregative stage

30 Capsaspora owczarzaki life cycle

C E

31 GO:0003700 sequence-specific DNA binding transcription factor activity (M) GO:0005768 endosome (C) GO:0005484 SNAP receptor activity (M) GO:0016236 macroautophagy (B) GO:0031201 SNARE complex (C) GO:0015991 ATP hydrolysis coupled proton transport (B) GO:0015144 carbohydrate transmembrane transporter activity (M) GO:0017156 calcium ion-dependent exocytosis (B) GO:0004713 protein tyrosine kinase activity (M) GO:0015629 actin cytoskeleton (C) GO:0003774 motor activity (M) GO:0006520 amino acid metabolic process (B) GO:0003779 actin binding (M) GO:0009062 fatty acid catabolic process (B) ribosome (GO:0005840) (C) GO:0005681 spliceosomal complex (C) histone acetylation (GO:0016573) (B) GO:0000398 mRNA splicing, via spliceosome (B) histone methyltransferase complex (GO:0035097) (C) GO:0007155 cell adhesion myosin complex (GO:0016459) (C) GO:0005874 microtubule (C) GO:0015991 ATP hydrolysis coupled proton transport (B) GO:0009060 aerobic respiration (B) GO:0005484 SNAP receptor activity (M) GO:0005657 replication fork (C) GO:0016236 macroautophagy (B) GO:0040010 positive regulation of growth rate (B) GO:0031201 SNARE complex (C) GO:0006261 DNA-dependent DNA replication (B) GO:0003700 sequence-specific DNA binding transcription factor activity (M) GO:0006270 DNA replication initiation (B) GO:0042575 DNA polymerase complex (C) GO:0006631 fatty acid metabolic process (B) GO:0046356 acetyl-CoA catabolic process (B) GO:0009083 branched-chain amino acid catabolic process (B) GO:0030055 cell-substrate junction (C) GO:0015629 actin cytoskeleton (C) GO:0015986 ATP synthesis coupled proton transport (B) GO:0005759 mitochondrial matrix (C) Floating vs Aggregative(1630/1466) GO:0003774 motor activity (M) GO:0045261 proton-transporting ATP synthase complex, catalytic core F(1) (C) GO:0005874 microtubule (C) GO:0009060 aerobic respiration (B) GO:0007091 mitotic metaphase/anaphase transition (B) GO:0046356 acetyl-CoA catabolic process (B) GO:0007076 mitotic chromosome condensation (B) GO:0051258 protein polymerization (B) GO:0030055 cell-substrate junction (C) GO:0005840 ribosome (C) GO:0003777 microtubule motor activity (M) GO:0006414 translational elongation (B) GO:0005925 focal adhesion (C) GO:0006270 DNA replication initiation (B) GO:0031105 septin complex (C) GO:0031012 extracellular matrix (C) GO:0034061 DNA polymerase activity (M) GO:0003700 sequence-specific DNA binding transcription factor activity (M) GO:0000084 S phase of mitotic cell cycle (B) GO:0004672 protein kinase activity (M) Floating vs all (1142/782/232) Aggregative vs all (233/166/0)

Floating vs Adherent (1711/1516)

Aggregative vs Adherent (679/675) GO:0015991 ATP hydrolysis coupled proton transport (B) GO:0005925 focal adhesion (C) GO:0031201 SNARE complex (C) GO:0043235 receptor complex (C) GO:0005085 guanyl-nucleotide exchange factor activity (M) GO:0007160 cell-matrix adhesion (B) GO:0008219 cell death (B) GO:0008092 cytoskeletal protein binding (M) GO:0005484 SNAP receptor activity (M) GO:0007229 integrin-mediated signaling pathway (B) GO:0003700 sequence-specific DNA binding transcription factor activity (M) GO:0007166 cell surface receptor linked signal transduction (B) GO:0006888 ER to Golgi vesicle-mediated transport (B) GO:0007267 cell-cell signaling (B) GO:0048193 Golgi vesicle transport (B) GO:0031012 extracellular matrix (C) GO:0016236 macroautophagy (B) GO:0005178 integrin binding (M) GO:0017157 regulation of exocytosis (B) GO:0004930 G-protein coupled receptor activity (M) GO:0005840 ribosome (C) GO:0003700 sequence-specific DNA binding transcription factor activity (M) GO:0006414 translational elongation (B) GO:0004713 protein tyrosine kinase activity (M) Adherent vs all (396/166/441) GO:0015629 actin cytoskeleton (C) GO:0007186 G-protein coupled receptor protein signaling pathway (B) GO:0046034 ATP metabolic process (B) GO:0006084 acetyl-CoA metabolic process (B) GO:0038023 signaling receptor activity (M) GO:0016459 myosin complex (C) GO:0004713 protein tyrosine kinase activity (M) GO:0003774 motor activity (M) GO:0008652 cellular amino acid biosynthetic process (B) GO:0005739 mitochondrion (C) GO:0003700 sequence-specific DNA binding transcription factor activity (M) GO:0006631 fatty acid metabolic process (B) GO:0006754 ATP biosynthetic process (B) GO:0008287 protein serine/threonine phosphatase complex (C) GO:0006270 DNA replication initiation (B) GO:0032147 activation of protein kinase activity (B) GO:0006323 DNA packaging (B) GO:0005765 lysosomal membrane (C) GO:0006493 protein O-linked glycosylation (B) GO:0004672 protein kinase activity (M) 32 Ichthyosporea: Sphaeroforma arctica

Jostensen et al. 2002 European Journal of Protistology 38:93-104. 33 Ichthyosporea: Sphaeroforma arctica

34 Ichthyosporea: Sphaeroforma arctica

34 transgenesis tools failed

35 Brachyury and T-box-rescue in Xenopus embryos in collaboration with José Luis Gómez-Skarmeta (Sevilla)

Control(MyoD)

Xbra-En (MyoD) 51,6% 41,9% 4,9%

Mutant Others phenotypes 1,6% Xbra-En + Xbra (MyoD) 13,6 % 64,07% 8,73%

Mutant +XenopusBrachyury

Xbra-En + Capsaspora A (MyoD) 15,94% 59,42% 23,1% Mutant +CapsasporaBrachyury

Xbra-En + Capsaspora B (MyoD) 6,06% 81,8% 12,12% Mutant +CapsasporaTbox3

36 Capsaspora Brachyury consensus binding-site (PBM, Protein Binding Microarray, in collaboration with Tim Hughes, Toronto)

37 Capsaspora Brachyury consensus binding-site (PBM, Protein Binding Microarray, in collaboration with Tim Hughes, Toronto)

Co Bra Mouse T Mouse Eomes

Mouse Tbx1 Mouse Tbx2 Mouse Tbx4

long-term goal: to perform ChipSeq

38 Capsaspora Hippo/Yap pathway (in collaboration with Doujia Pan, Howard Hughes Medical Institute, Baltimore)

Sebé-Pedrós et al. 2012. Cell Reports doi:10.1016/j.cel- rep.2011.11.004 39 Capsaspora Hippo/Yap pathway (in collaboration with Doujia Pan, Howard Hughes Medical Institute, Baltimore)

Sebé-Pedrós et al. 2012. Cell Reports doi:10.1016/j.cel- rep.2011.11.004 39 Our new baby, the ichthyosporean Creolimax fragrantissima

Marshall et al. 2008. Protist 159:415-433.

40 Our new baby, the ichthyosporean Creolimax fragrantissima

41 First transformation in an unicellular relative of Metazoa!!!!!

beta-tubulin-promoter beta-tubulin- Creolimax+S.arctica-histone promoter Creolimax +mcherry +venus

42 First transformation in an unicellular relative of Metazoa!!!!!

beta-tubulin-promoter beta-tubulin- Creolimax+S.arctica-histone promoter Creolimax +mcherry +venus

42 First transformation in an unicellular relative of Metazoa!!!!!

beta-tubulin-promoter beta-tubulin- Creolimax+S.arctica-histone promoter Creolimax +mcherry +venus

43 Capsaspora owczarzaki

44 and three months ago...

45 more genomes?

Fungi

Corallochytrium limacisporum Choanoﬂagellata Nucleariidae Salpingoeca Monosiga F. alba ? Filasterea rosetta brevicollis Metazoa Capsaspora Ministeria owczarzaki vibrans

Ichthyosporea Sphaeroforma arctica Amoebidium parasiticum

Creolimax fragrantissima

T. trahens Apusozoa

46 single-cell genomics2,("%(/5"1&55"+&" "2"%+,(&2$8" "2"%+,(&2"

*+&),"%(&2"

8$"),"%(&2"

!"2,('3 ?

"2"%+,(2(#4$*

!(%(*$1" 2#"*)&8$8"

5"%1&%(&2"

? /#&*2,('3

0#2&(5"+"

*"5)$%1(&2"

%")$/(#!$*

? /#&*2,('6 )9:$8$0!

? )0+"+$>& /#&*2,('7 2,("%(/5"1&55"+&* ?

25"8&'5 ?

!"()'6 !"#$%&' ? ()$*+,(-(%+" !"()'3 ? 2(#"55(2,9+#$0! $2,+,9(*)(#&" $2,+,9(*)(%$8" &22#$%"5&*

$2,+,9(*)(%0*

"!(&4$8$0!

/#&*,$)'3 ?

)*(#(*)&#!$0!

"4&(/(#!"

!"$)'3 ?

"%0#(/&2"

)*&08()&#-$%*0*

8&#!(29*+$8$0! 8&#!(29*+$8"

2")*"*)(#" /$5"*+&#&" !&+".(" 47

!$%$*+&#$" /$5"*+&#&" 8$*2$2#$*+($8&" *$!)5&: %025&"#$$8"

+,&#!(),$5"

/(%+$205" /(%+$205$8&"

/#&*,@"+&# ? /#&*,() ()$*+,(-(%+" /0%1$

(0+1#(0)

;<;= Acknowledgments

http://www.multicellgenome.com

Our collaborators: Broad Institute team, Franz Lang, Andrew J. Roger, Nicole King, Kamran Shalchian- Tabrizi, Maja Adamska, Todd Miller, Gerard Manning, Jose Luis Gómez-Skarmeta, Doujia Pan.

ARCELONAAT