Report individual group leaders

Site visit Hubrecht Institute 10-11 November 2014

- 2 - INDEX

Group leader Group name Page

Jeroen Bakkers Cardiac Development and Genetics ...... 05

Hans Clevers Lgr5 stem cells, Wnt signaling & cancer ...... 11

Menno Creyghton Neuro epigenetics ...... 19

Edwin Cuppen Genome Biology and Medical Genetics ...... 25

Eelco de Koning Diabetes and islet neogenesis ...... 33

Wouter de Laat Biomedical Genomics ...... 39

Jeroen den Hertog Protein-tyrosine phosphatases in development ...... 47

Jacqueline Deschamps Genetics of morphogenesis during axial elongation in the mouse embryo ...... 53

Niels Geijsen Stem Cell Modeling of human genetic disease ...... 61

Daniele Guardavaccaro Ubiquitin ligases and cancer ...... 67

Jop Kind Spatiotemporal regulation of genomic function ...... 73

Puck Knipscheer Molecular mechanisms and regulation of DNA repair...... 77

Rik Korswagen Wnt signaling in development and disease ...... 83

Catherine Rabouille Secretion regulation ...... 89

Catherine Robin Hematopoiesis and stem cells during embryonic development ...... 95

Alexander van Oudenaarden Quantitative biology of development & stem cells ...... 103

Jacco van Rheenen Cancer Biophysics ...... 113

Eva van Rooij Molecular Cardiology ...... 121

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Jeroen Bakkers

Key publications (2008-2014)

Junker JP, Noël ES, Guryev V, Peterson KA, Shah G, Huisken J, McMahon AP, Berezikov E, Bakkers J* and van Oudenaarden A*. Genome-wide RNA tomography in the zebrafish embryo. *shared corresponding authors. Cell in press.

Chetaille P, Côté J, Burkhard S, Houde C, Preuss C, Piché J, Gosset N, Leclerc S, Wünnemann F, Cameron M, Castilloux J, Thibeault M, Gagnon C, Galli A, Tuck A, Hickson G, Amine N, Boufaied I, Lemyre E, Santa Barbara P, Faure S, Jonzon A, Dietz H, Gallo-McFarlane E, Benson W, Zhan SH, Shen Y, Jomphe M, Jones SJM, Bakkers J and Andelfinger G. A human dysrhythmia syndrome affecting heart and gut is associated with mutations in SGOL1. Nat. Genet. in press.

Noël ES, Verhoeven M, Lagendijk AK, Tessadori F, Smith K, Choorapoikayil S, den Hertog J, Bakkers J. (2013) A Nodal-independent and tissue-intrinsic mechanism controls heart-looping chirality. Nat. Commun. 4:2754.

Mugoni V, Postel R, Catanzaro V, De Luca E, Digilio G, Turco E, Silengo L, Murphy MP, Medana C, Stainier DYR, Bakkers J and Santoro MM. (2013) Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis. Cell 152(3):504-518.

Smith K, Chocron S, von der Hardt S, de Pater E, Soufan AT, Bussmann J, Schulte Merker S, Hammerschmidt M and Bakkers J. (2008). Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells. Dev. Cell 14; 287–297.

- 5 - Dr. ir. Jeroen Bakkers Cardiac Development and Genetics

Group members

Postdocs: Emily Noël, Federico Tessadori, Ina Strate Graduate students: Lotte Koopman, Silja Burkhard, Fabian Kruse, Melanie Laarman Technicians: Sonja Chocron

Curriculum vitae group leader

Name: Dr. ir. Jeroen Bakkers Date of birth: 13-05-1970 Nationality: Dutch

Education/positions 1988-1994 MSc, Wageningen University 1994-2000 PhD, Leiden University 2000-2003 Postdoc, Max-Planck Institute, Freiburg, Germany

2003-2008 Hubrecht Institute, Junior group leader 2008-now Hubrecht Institute, Senior group leader

Memberships - Member editorial board Journal of Cardiovascular Development and Disease - President of the Dutch Society of Developmental Biology (DSDB).

Awards - 2000 PhD degree with highest distinction (Cum Laude), Leiden University - 2009 NWO/Vidi career grant

Other activities • Organizer meetings: 2008, 2011, 2013 National meeting on Cardiac Development, Utrecht. 2008 KNAW colloquium “Cardiac Development, Disease and Stem Cells”, Amsterdam. 2011 1st meeting of the Dutch Society for Developmental Biology (DSDB), Utrecht.

Thesis advisor for (graduation date) • Ruben Postel, (May 2008) • Manon Verhoeven, (April 2009) • Emma de Pater, (June 2010) • Anne K. Lagendijk, (December 2011)

Invited speaker on meetings (2008-2014) 2008: Weinstein meeting for Cardiovascular Development, Houston, USA 2009: American Heart Association (AHA) Scientific Sessions, Orlando, USA; European Heart Failure meeting (ECS), Nice, France; Joint Meeting 2009 Anatomische Gesellschaft – Nederlandse Anatomen Vereniging, Antwerpen, Belgium; Dutch-German meeting on Cardiovascular research, Hamburg, Germany; Zebrafish PI meeting, Asilomar, USA 2010: EUGeneHeart Symposium, Brussels, Belgium; Basic Cardiovascular Science Meeting of the European Society of Cardiology, Berlin, Germany; Heart Failure Congress (ESC), Berlin, Germany 2011: International Weinstein meeting for Cardiovascular Development, Cincinnati, USA; Zebrafish PI meeting, Asilomar, USA 2012: International Weinstein meeting for Cardiovascular Development, Chicago, USA; Basic Cardiovascular Science Meeting of the European Society of Cardiology, London, UK; ESC Working Group Cardiac Development Meeting, Amsterdam, NL; International meeting on BMP signaling in Development and Disease, Lake Tahoe, USA

- 6 - 2013: Zebrafish PI meeting, Asilomar, USA; Zebrafish Development and Disease meeting, Bristol, UK 2014: Zebrafish Disease Models Conference, Madison, USA; Graduate School Student Meeting Münster, Germany; CDBC Spring symposium, Charleston, USA

Grants (2008-2014) • 2014 NWO middelgroot (equipment), co-PI € 550,000 • 2013 Nederlandse Hartstichting, co-PI € 250,000 • 2013 ZonMW TOP grant, PI € 290,000 • 2013 CVON CardioVasculair Onderzoek Nederland, co-PI € 200,000 • 2012 CVON CardioVasculair Onderzoek Nederland, co-PI € 450,000 • 2012 NWO PhD fellowship, to S. Burkhard € 200,000 • 2011 NWO ZonMW middelgroot, co-PI € 400,000 • 2009 EU FP7 BIOSCENT, co-PI € 250,000 • 2009 NWO Vernieuwingsimpuls (VIDI), career grant), PI € 600,000 Total: € 3,200,000

Previous research

My research is centred on understanding mechanisms of cardiac development and disease using the zebrafish as a model system. During my postdoctoral training I had worked on the role of bone morphogentic protein (BMP), a TGF-ß related growth factor, during gastrulation. I had worked with several zebrafish mutants deficient in BMP signalling components and that displayed cardiac phenotypes that had not been described previously. Considering the advantages of the zebrafish model (good genetic tools, rapid development and transparency of the embryo, and early development independent of functional cardiovascular system), and the early lethality of mouse mutants defective in BMP signalling made me decide to study the role of BMP signalling in cardiac development when starting my own research group at the Hubrecht Institute. This initial work let to the discovery that BMP signalling is required at post-gastrula stages to regulate the asymmetric development of the heart (Chocron et al. (2007) Dev. Biol.). During heart tube formation the inflow pole of the linear heart tube will be positioned left from the embryonic midline after which it forms an S-shaped heart tube with the ventricle moving towards the embryonic right side. Our work demonstrated that BMP signalling is required at two different stages to regulate the asymmetric development of the heart tube; 1) BMP signalling is required to establish correct expression of early laterality markers (such as left-sided Nodal expression) during early somite stages. 2) During late somite stages when asymmetric Nodal expression is established BMP signalling is required for the leftward positioning of the heart tube. To reveal the cell migration behaviour of cardiac progenitor cells during asymmetric heart tube formation I combined confocal time-lapse microscopy with cell tracking tools. Doing so we discovered an asymmetric cell behaviour that results in the clock-wise rotation of the cardiac field during heart tube formation. In addition we found that BMP growth factors can influence cell migration behaviour and regulate the direction of heart tube assembly (Smith et al (2008) Dev. Cell). Other labs have independently confirmed this rotation behaviour and demonstrated that also Nodal growth factors influence migration behaviour of cardiac progenitor cells suggesting a close relation of BMP and Nodal signalling during this process.

To allow an unbiased approach to identify components that regulate the left-right axis and/or asymmetric organ development my lab performed several ENU-based forward genetic screens that used alterations in heart laterality (leftward position and directional heart looping) as a read-out (excluding mutants with overt cilia related defects). We screened around 1200 F2 families (about 100.000 embryos) over a period of 2-3 years resulting in the validation of 10 independent mutant lines. Interestingly we found that these mutant lines fall into two different classes; Class I, affecting left-right axis formation and laterality of all organs: Class II, affecting heart looping without affecting laterality of other organs. Thus far we have mapped and identified the causing mutation of 5 of these mutant lines using classical mapping approaches and sequencing candidate genes in the genomic region. Characterization of these mutants resulted in several publications (Noël et al (2013) Nat. Commun.; Smith et al (2011) PLoS Genet.; Smith et al (2011) Development; Tessadori et al. Dev. Cell in revision). Main conclusions from this published and unpublished work are: 1) BMP signalling represses Nodal signalling during LR patterning by driving the expression of the Nodal-antagonist Lefty1 in the midline. 2) The signalling range of Nodal in the lateral plate mesoderm is regulated by Furin mediated cleavage. 3) Directional heart looping is regulated by Nodal-dependent and –

- 7 - independent mechanisms. 4) The novel transmembrane spanning protein Tmem2 is required for cardiac looping and restricting cardiac valve formation. I will continue to work on the remaining mutants (5) (see below).

In 2001 several labs published that in mouse and chick embryos the linear heart tube growths at both poles by the addition of new cardiomyocytes that originate from the pharyngeal mesoderm. The cardiac progenitor cells giving rise to these late cardiomyocytes were named second heart field cells. Tracing experiments revealed that second heart field cells give rise to the right ventricle and parts of both atria. Since fish don’t have a right ventricle (only have a single ventricle) and have only one atrium it was speculated that the second heart field is specific to amniotes to establish addition cardiac chambers that form the pulmonary circulation system. However, results from my lab demonstrated that also the fish heart growths by the addition of new cardiomyocytes at the poles of the heart tube (de Pater et al (2009) Development). Other research groups that acknowledge our initial finding later confirmed the presence of a second heart field in fish. We continued to investigate the regulation of cardiomyocyte differentiation from the second heart field and found that BMP signalling has a dual role in this; 1) BMP signalling is required to specify cardiac progenitor cells and induces the expression of the main transcription factor Nkx2.5. 2) BMP signalling needs to be repressed in cardiac progenitor cells to allow efficient cardiomyocyte differentiation (de Pater et al (2011) Circ. Res.; Strate et al. Development in revision). Repression of BMP signalling requires Smad6 and Glypican4.

To identify novel genes that are expressed in a specific pattern in the zebrafish embryo (left-right asymmetry, heart field) I collaborated with Alexander van Oudenaarden to develop a new technique that we named RNA tomography (Junker et al. Cell in press). This technique is based on sectioning of a whole embryo in thin (15 μm) slices and extracting and sequencing RNA from individual slices (tomo-seq). Tomo-seq is quantitative and reproducible and we succeeded in resolving known and unknown expression patterns (confirmed by in situ hybridization). By sectioning zebrafish embryos in 3 different orientations (anterior-posterior, dorsal-ventral, left-right) and mapping the tomo-seq data on a 3D reference embryo we generated genome-wide RNA expression patterns that can be visualized in a virtual embryo. Thus far we performed this for embryos of 3 different developmental stages and the data will be available to the research community through a searchable database. The main advantage of this technique is that it allows to search for genes with expression patterns similar to a gene of interest to identify uncharacterized candidate genes that could be important for a specific developmental process (e.g. left-right patterning, cardiac progenitor cell specification/differentiation).

Cardiac valves, that are required to prevent the blood from flowing back from the ventricle in the atrium, are initiated when the heart tube starts to loop. Studying maternal zygotic dicer mutant embryos we discovered that cardiac looping and valve formation requires Dicer activity. By a knock- down strategy we identified microRNA-23 to be essential for valve formation by repressing the activity of the extracelluar matrix producing enzyme hyaluronan synthase 2, and thereby restricts valve formation to the atrioventricular canal (Lagendijk et al (2011) Circ Res.). Defective valve formation results in serious congenital heart defects in human patients. Earlier work had suggested that a genetic component is involved in causing these congenital valve malformations. To address this more directly I established collaborations with cardiologists Barbara Mulder (Amsterdam Medical Center) and Edwin Cuppen (HI) to sequence candidate genes derived from literature on DNA samples from patients with congenital valve defects. Sequencing 30 candidate genes in 200 patient samples and 300 control samples using Sanger sequencing revealed 10 genes with putative disease causing mutations. Corroborating the hypothesis that cardiac valve malformation can be caused my genetic defects we found by in vitro and in vivo analysis of 2 of these genes that the patient specific mutations make the protein inactive or dominant negative (Smith et al (2009) Circulation; Hyde et al (2012) JBC).

The electrical signal (membrane depolarization) that initiates cardiac contraction is generated in the pacemaker cells of the sinoatrial node located at the base of the right atrium and the sinus venosus. Although pacemaker activity had been identified and located in the sinoatrial region of the fish heart, no cellular- or molecular markers had been described to identify the pacemaker cells. We discovered by serendipity that islet-1 mutant embryos display cardiac arrhythmias that could be related to pacemaker disfunction (de Pater et al (2009) Development). By a transgenic approach using BAC recombination and electrophysiology on single cells we found that Islet-1 expressing cells of the zebrafish heart have pacemaker activity and Islet-1 expression in pacemaker cells is conserved in mouse and human (Tessadori et al (2012) PLoS One). Due to this initial work on zebrafish pacemaker

- 8 - cell development Gregor Andelfinger, a cardiologist located in Montreal Canada, contacted me to collaborate on a novel human syndrome that involves simultaneous defective gut and heart pacemaker activity. His group had performed whole-exome sequencing and identified a homozygous mutation in all patients (14) in the cohesin complex component shugoshin-like 1 (SGOL1) gene that resulted in a change of a highly conserved amino acid SGOL1 (c.67 A>G [p.Lys23Glu]. My group identified a zebrafish sgol1 homologue that is expressed in the embryonic heart and gut and in the sinoatrial node cells of the adult heart. We performed loss-of-function studies for Sgol1 in zebrafish embryos using a knock-down approach and demonstrated that Sgol1 is required to maintain cardiac rhythm in the embryonic heart (Chetaille et al. Nat. Genet. in press).

Future research

I will continue to investigate the molecular mechanism that regulate left-right axis formation and determine asymmetric morphogenesis of the heart. From the ENU-based forward genetic screen there are 5 uncharacterized mutants left that display defects in left-right patterning or heart morphogenesis. The causative mutations will be mapped and identified using whole exome-sequencing in collaboration with Kelly Smith (former postdoc and now independent group leader at the IMB, Brisbane, Australia). Furthermore from the tomo-seq data that we have available we identified new genes that are asymmetrically expressed in relation to the midline. We identified 10 genes that are enriched in the left side of the embryo of which 2 are known left-right genes (spaw and cyclops) and 8 are uncharacterized genes. In addition we identified 2 genes that are enriched in the right side of the embryo which are uncharacterized thus far. New genome-engineering tools such as CRISPR/Cas will be used to generate loss-of-function models and investigate their function in left-right patterning. To determine conservation of left-right patterning across species we will perform tomo-seq on embryos of various species such as mouse, chick, Ciona intestinalis (in collaboration with Brad Davidson, Swarthmore College, USA) and pig (in collaboration with Bernard Roelen, Utrecht University). In our recent published work we demonstrate that in the absence of Nodal signaling there is asymmetric morphogenesis of the heart, which requires actin polymerization and myosin activity (Noël et al (2013) Nat. Commun.). I hypothesize that there could be similarity with Drosophila and C. elegans since these species lack Nodal factors and require actomyosin activity for asymmetric (organ) morphogenesis. We are using transgenic approaches (LifeAct, Myosin-GFP) to visualize actin and non-muscle myosin dynamics in in vivo in the zebrafish heart. In addition these transgenes will allow functional analysis by laser-ablation in the future. Furthermore we will perform timelapse imaging of the looping process to visualize the simultaneous growth and looping of the heart tube and determine how growth affects this process using mutants defective in this process. I have established collaborations with Carlijn Bouten and Frank Baas (Biomedical Engineering, Technical University Eindhoven) to perform physical measurements (e.g. stiffness) on embryonic heart tissue and model the looping process using mathematical modeling.

My lab demonstrated that the transcription factor Islet-1 marks cardiac pacemaker cells in zebrafish, mouse and human and that its activity is required for pacemaker function. I will continue to study the role of Islet-1 in pacemaker development using the islet-1 mutant and the transgenic lines we created (islet-1BAC:Gal4). I will further develop the tomo-seq protocol so that we can perform tomo-seq experiments on isolated heart tissue and identify other genes specifically expressed in the pacemaker cells (located at the inflow pole) and of which the expression is affected in the islet-1 mutant. We found that another transcription factor (Tbx2) is downstream of Islet-1 and in collaboration with Vincent Christoffels (Amsterdam Medical Center), who is an expert in Tbx2 and Tbx3 function in mouse heart development, I will study the regulation of pacemaker cell differentiation by these transcription factors. Since human patients with SGOL1 mutation have pacemaker dysfunction I will use the CRISPR/Cas genome-engineering tool to create zebrafish knock-out alleles for sgol1. I have successfully extended CRISPR-Cas technology with homologous recombination to introduce patient-derived genetic variants into the zebrafish genome. I will use this approach to create a zebrafish knock-in line with the patient- derived sgol1 23K>E variant. Cardiac function of mutant larval hearts will be analyzed using established and new methods (e.g. FRET sensors for membrane potential and calcium concentrations in collaboration with Teun de Boer, Medical Physiology UMC Utrecht). Since Sgol1 is a cohesion- binding factor I would like to test the hypothesis that loss of Sgol1 may result in differences in chromatin organisation and transcriptional regulation (in collaboration with Wouter de Laat (HI) and Menno Creyghton (HI)).

- 9 - Zebrafish have the unique ability that they are able to regenerate their heart to restore heart function after damage. Many developmental genes are differentially expressed in the regenerating adult heart. I established a cryo-injury protocol to damage the heart of adult zebrafish to study the role for BMP signalling in the regeneration process (in collaboration with Gilbert Weidinger, University of Ulm, Germany). By using transgenic approaches to manipulate BMP signalling and using a BMP receptor mutant that is adult viable we have preliminary data indicating that BMP signalling is required for the early response that regulates cardiomyocyte proliferation. I will use the tomo-seq approach to investigate where BMP signalling is activated in the regenerating heart and identify genes of which the expression is regulated by the BMP response. Furthermore the tomo-seq method will be applied to injured mouse hearts (in collaboration with Eva van Rooij (HI) and Vincent Christoffels (AMC) and injured mouse neonatal hearts (in collaboration with Paul Riley, Oxford, UK) to identify (BMP related) pathways that are differentially regulated in regenerating and non-regenerating hearts. We observed that BMP signalling is activated in proliferating cardiomyocytes. I want to extend the tomo-seq protocol to do single cell RNA-seq to compare BMP responsive versus BMP non-responsive cardiomyocytes and identify BMP regulated pathways important for cardiomyocyte proliferation.

Societal relevance and societal impact (2008-2014)

Cardiovascular disease is the leading cause of death in the western world costing approximately €315 billion annually including health expenditures and lost productivity that results from premature mortality. Recent developments in exome-sequencing and genome-wide association studies have illuminated a spectrum of genetic underpinnings linked to several cardiovascular diseases. It is very important to determine whether there is an underlying genetic basis for the disease phenotype from an economic and societal perspective, as it would save on other diagnostic activities, which are burdens on both the family’s involved and healthcare budgets. Similarly, patients typically require life-long intensive treatment or care from both professionals and parents. Understanding the underlying genetic causes will give insight into the pathobiological basis of the disease, allow definition of disease risk and progression, and identify therapeutic targets.

Genome-wide association studies (GWAS) reveal a remarkable genetic complexity of cardiovascular diseases. Recent analysis of GWAS data sets has demonstrated that common variants associated with phenotypic outcomes are most often located in non-coding regions of the DNA that have a role in regulating gene expression. Furthermore these regulatory sequences are active only during embryonic development or during embryonic development and postnatal stages, indicating that disease-causing genes are required during organ development in the embryo. However the role of these regulatory regions and the role of the genes that they interact with during organ development is in most cases unknown. Therefore extensive knowledge of the genes, their regulation and their molecular mechanisms underlying normal cardiovascular development is required. Furthermore the knowledge on genes and mechanisms that drive conduction system formation are not only beneficial to understand cardiac rhythm diseases but are also beneficial to regenerative medicine projects. I coordinate a consortium with Vincent Christoffels (AMC) and Christine Mummery (Leiden UMC) to develop tools for making biological pacemakers in vitro, which will be greatly benefit from the extensive knowledge generated here about the factors and mechanism driving pacemaker cell differentiation in vivo.

The genome-wide RNA tomography technique that we developed is applicable to other systems. We have had many requests from other researchers that want to use the tomo-seq technique to address biological questions in other organisms and organs. Besides the technological advance, RNA tomography results will be very useful to other researchers working on zebrafish. We will make all data publically available in a database that can be easily searched for expression patterns and genes with expression pattern similar to a gene of interest. Furthermore we will link the data with existing databases such as ZFIN to make the data available to the entire zebrafish research community.

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Hans Clevers

Key publications (2008-2014)

Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M, Danenberg E, Clarke AR, Sansom OJ, Clevers H. (2009) Crypt Stem Cells as the Cells-of-Origin of Intestinal Cancer. Nature 457(7229):608-11.

Sato T, Vries R, Snippert H, van de Wetering M, Barker N, Stange D, van Es J, Abo A, Kujala P, Peters P and Clevers H. (2009) Single lgr5 gut stem cells build crypt-villus structures in vitro without a stromal niche. Nature 459(7244):262-5.

Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, Clevers H. (2010) Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469(7330):415-418.

Snippert J, van der Flier LG, Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein AM, van Rheenen J, Benjamin D, Simons BD, Clevers H. (2010) Intestinal Crypt Homeostasis results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells. Cell 143(1):134-44. de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M, Stange DE, van Es JE, Guardavaccaro D, Schasfoort RB, Mohri Y, Nishimori K, Mohammed S, Heck AJ, Clevers H. (2011) Lgr5 homologues associate with Wnt receptors and mediate R-spondin signaling. Nature 476(7360):293-7.

Koo B-K, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M, van Es JH, Mohammed S, Heck AJR, Maurice MM, Clevers H. (2012) Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488(7413):665-9.

Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H. (2012) Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas. Science 337(6095):730-5.

Huch M, Dorell C, Boj SF, van Es JH, van de Wetering M, Li VSW, Hamer K, Sasaki N, Finegold MJ, Haft A, Grompe M, Clevers H. (2013) In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494(7436):247-50.

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Prof. Dr. Hans Clevers Lgr5 stem cells, Wnt signaling & cancer

Group members

Scientists: Marc van de Wetering, Johan van Es, Wim de Lau, Robert Vries Postdocs: Sina Bartfeld, Onur Basak, Sylvia Boj, Jarno Drost, Henner Farin, Helmut Gehart, Inha Heo, Oded Kopper, Kai Kretschmar, Norman Sachs, Nobuo Sasaki, Valentina Sasselli, Gerald Schwank Graduate students: Paul Tetteh Technicians: Tulay Bayram, Harry Begthel, Joyce Blokker, Maaike van den Born, Stieneke van den Brink, Sepideh Derakhshan, Ana Gracanin, Benaissa el Haddouti, Karien Hamer, Sridevi Jaksani, Jeroen Korving, Tamana Mehraban, Marvin Statia, Margriet Westerveld, Laura Zeinstra

Curriculum vitae group leader

Name: Johannes Carolus Clevers Date of birth: March 27, 1957 Nationality: Dutch

Education/positions 1982 M.Sc. in Biology, University of Utrecht 1984 M.D., University of Utrecht 1985 Ph.D., University of Utrecht

1985 - 1989 Research Fellow in Pathology. Dana-Farber Cancer Institute, Harvard Medical School, Boston MA, USA 1989 - 1991 Universitair Docent, Department of Clinical Immunology,University of Utrecht 1991 - 2002 Professor and Chairman, Dept. of Immunology, Faculty of Medicine, University of Utrecht 2002 - 2012 Director of Hubrecht Institute, Developmental Biology and Stem Cell Research 2002 Professor, Molecular Genetics, University of Utrecht 2002 Honorary professor of Central South University of Changsha (Hunan), China 2012 President of the Royal Netherlands Academy of Arts and Sciences (KNAW)

Memberships 1999 Member European Molecular Biology Organisation (EMBO) 2000 Member of the Royal Netherlands Academy of Sciences 2004 Member of the International Society for Stem Cell Research (ISSCR) 2005 Member of the Scientific Advisory Board of ISREC, Swiss Institute for Experimental Cancer Research 2006 Member Scientific Committee of the Louis-Jeantet Foundation for Medicine 2006 Member of the American Association for Cancer Research (AACR) 2007 Member of the National Scientific Advisory Board NKI-AVL 2008 Member of the scientific advisory board of “de Anatomische Les” 2009 Member of the Academia Europaea 2010 Member of the Scientific Advisory Board of the MRC Clinical Sciences Centre 2012 Member of the American Academy of Arts and Sciences 2012 Member of the ‘Koninklijke Hollandsche Maatschappij der Wetenschappen’ (The Royal Holland Society of Sciences and Humanities) 2012 Member of the Gairdner Foundation medical advisory board 2014 Foreign Associate of the US National Academy of Sciences

Editorial boards 2004 Member of the Editorial Board of the EMBO Journal 2007 Member of the of the Editorial Board of Gastroenterology 2008 Member of the Editorial Board of the Journal: Diseases, Models and Mechanisms 2009 Member of the Editorial Board of Cell 2010 Member of the Editorial Board of Genes & Development 2011 Member of the Editorial Board of Gastroenterology Report

- 12 - 2012 Member of the Editorial Board of Stem Cell Reports 2013 Member of the Editorial Board of Cell Stem Cell

Awards 2000 Catharijne-prize for medical science 2001 Award from the European Society for Clinical Investigation 2001 Spinoza-award 2004 Louis-Jeantet Prize for Medicine 2005 the Science and Society Prize 2005 the French honor of “Chevalier de la Legion d'Honneur” 2005 Katharine Berkan Judd Award 2006 Rabbi Shai Shacknai Memorial Prize for Immunology and Cancer Research 2008 Josephine Nefkens Prize for Cancer Research (Erasmus MC, Rotterdam) 2008 Meyenburg Cancer Research Award 2009 The Queen Wilhelmina Dutch Cancer Society Award 2010 The United European Gastroenterology Federation (UEGF) Research Prize 2011 The Ernst Jung Medical Award 2011 Kolff prize 2012 Association pour la Recherche sur le Cancer (ARC) Léopold Griffuel Prize 2012 William Beaumont prize of the American Gastroenterology Association 2012 The Heineken Prize for Medicine 2012 Ridder in de Orde van de Nederlandse Leeuw 2013 The Breakthrough Prize in Life Sciences 2014 Massachusetts General Hospital Award in Cancer Research 2014 TEFAF Oncology Chair 2014 2014 Fellow of the AACR Academy 2014 Struyvenberg European Society for Clinical Investigation (ESCI) medal

Thesis advisor 30 PhD students have graduated under my direct supervision in the past five years, 8 of which in my own research group: 2009 Laurens van der Flier 2010 Ana Faro 2010 Sue Ng 2011 Hugo Snippert 2012 Daniel Stange 2012 Arnout Schepers 2013 Jurian Schuijers 2013 Wouter Karthaus

Invited speaker on meetings (2008-2014) I give 70-80 talks per year. Below are the Keynotes and Named Lectures from 2008-2014. • Keynote Lecture at the Dutch-German meeting of Molecular Cardiology groups 2008, Amsterdam (February 7, 2008) • Keynote Lecture at the European Multidisciplinary Colorectal Cancer Congress 2008, Berlin (February 24, 2008) • Keynote Lecture at the 2008 Keystone Symposium on Signaling Pathways in Cancer and Development, Steamboat Springs, Colorado (March 24, 2008) • Keynote lecture at the Giovanni Armenise-Harvard Foundation 12th annual symposium Stresa, Italy (June 20, 2008) • PCDI (Postdoc Career Development Initiative) Retreat, Heeze, the Netherlands (November 7, 2008) • Keynote lecture at the FASEB Summer Research Conference entitled: Gastrointestinal Tract XIII: Advances in the Molecular & Cell Biology of the Intestinal Epithelium: Development, Inflammation, Host Defense & Cancer, Snowmass Village, CO (August 9, 2009) • Keynote lecture at the EMBO Molecular Medicine Workshop “Invasive Growth: a Genetic Programme for Stem Cells and Cancer” in Turin (September 12, 2009.) • Keynote lecture at Biannual Meeting on Stem Cells, Cold Spring Harbor Laboratory (September 25, 2009) • Keynote lecture at BioValley Life Sciences Week / MipTec Conference (October 13, 2009)

- 13 - • Leukaemia Research Annual Guest Lecture, London (November 11, 2009) • Keynote lecture at James Watson Cancer Symposium, Suzhou, China (April 8, 2010) • David de Wied-lezing, University Utrecht (May 28, 2010) • Keynote lecture International Student Congress of Medical Sciences (ISCOMS), University Medical Center Groningen (June 9, 2010) • Keynote lecture at the 5th lustrum of the training of the Biomedical Sciences of the Utrecht University (June 11, 2010) • Keynote lecture at the Falk symposium: IBD Benelux Summit 2010, Maastricht (June 25, 2010) • Sir Richard Gardner Celebratory Lecture, University of Scotland (November 5, 2010) • Keynote lecture at Fortaleza Conference (Brasil), December 7, 2010 • Keynote lecture at Tornado WP3-WP4 meeting: Exploring the interaction between man and microbiata, Lausanne, January 24, 2011 • Max Birnstiel lecture at the Research Institute of MolecularPathology, Vienna (February 2, 2011) • Keynote lecture at the 7th annual Swiss Stem Cell Network meeting, Lausanne (February 4, 2011) • Streisinger Lecture at the University of Oregon (February 22, 2011) • Mendel Lecture, Brno (May 12, 2011) • Keynote lecture at the 97th Annual Meeting of the Japanese Society of Gastroenterology, Tokyo (May 15, 2011) • Keynote lecture at the COE symposium at the Keio University, Tokyo (May 16, 2011) • Keynote lecture at the BMM/TeRM Annual Meeting 2011, Ermelo (May 25, 2011) • Keynote Lecture at the FASEB Summer Research Conference: Gastrointestinal Tract XIV: Stem Cells, Adaptation, Inflammation and Cancer (August 14, 2011) • The Weigle lecture at the University of Geneva (October 18, 2011) • Keynote lecture at the joined meeting of the Dutch endothelial Biology Society and the Dutch Cell Biology Society, KNAW Amsterdam (October 21, 2011) • Plenary talk at EuroSystem-Advances in Stem Cell Research VI Conference, Kranjska Gora, Slovenia (January 23, 2012) • Keynote lecture at the 16th Molecular Medicine Day, Rotterdam (February 29, 2012) • Keynote Lecture at the Keystone Symposium: The Life of a Stem Cell: From Birth to Death, Olympic Valley, CA (March 11, 2012) • Provost's Lecture, MD Anderson Cancer Center (March 16, 2012) • Keynote Lecture at the 24th Annual Meeting of the European Renal Cell Study Group, Doorwerth, the Netherlands (March 22, 2012) • Keynote Lecture at the Abcam Stem Cell Symposium, Boston (April 30, 2012) • Keynote Lecture at the 3rd EMBO Conference Series on Cellular Signaling & Molecular Medicine, Dubrovnik (May 28, 2012) • Mühlbock Memorial Lecture at the 22nd Biennial Congress of the European Association for Cancer Research (EACR), Barcelona (July 7, 2012) • Keynote Lecture at the Cell Tumour Microenvironment Lablinks symposium, London (July 9, 2012) • Keynote Lecture at the Heinrich F. C. Behr-Symposium, Heidelberg (October 16, 2012) • Keynote Lecture at the Food Valley Expo, Arnhem (October 25, 2012) • Jean Brachet Memorial Lecture at the International Society of Differentiation Conference "Stem Cells, Development and Regulation, Amsterdam (November 6, 2012) • Keynote lecture at the Student Research Conference 2012, Utrecht (November 21, 2012) • van Leeuwenhoek Lecture on BioScience, Leiden (February 28, 2013) • Keynote lecture at the 8th Cancer Scientific Forum of the Cancéropôle CLARA, Lyon (March 21, 2013) • The opening plenary lecture at the AACR Annual meeting, Washington (April 7, 2013) • CeMM Landsteiner Lecture, Vienna (May 6, 2013) • Methusalem Lecture, Leuven (May 24, 2013) • EMBO Keynote Lecture at FASEB SRC "The TGF-B Superfamily: Signaling in Development & Diseases", Steamboat Springs, Colorado (July 28, 2013) • Keynote lecture at the AACR Third International Conference on Frontiers in Basic Cancer Research 2013, Washington (September 18, 2013) • State of the Art Lecture at the anniversary conference of the Nederlandse Vereniging voor Gastroenterologie, Veldhoven (October 3, 2013) • Keynote at the 2013 European Cancer Congress, Amsterdam (September 28, 2013)

- 14 - • Keynote Lecture at the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, Boston (October 20, 2013) • Plenary lecture at the 2014 FISEB (Federation of the Israeli Societies for Experimental Biology) Conference, Eilat (February 12, 2014) • Plenary lecture at the satellite symposium Regenerative medicine, Utrecht (May 2, 2014) • Plenary Lecture at the 2014 annual meeting of the Japanese Society of Developmental Biologists, Nagoya, Japan (May 29, 2014) • Keynote lecture at the European Molecular Imaging Meeting (EMIM 2014), Antwerp (June 4, 2014) • Keynote lecture at the 26th Pezcoller Symposium, Trento, Italy (June 19, 2014)

Grants (2008-2014) Grant Start Amount (€) Cancer Genomics center II 2008 1,500,000 KWF award (KWF/PF-HUBR 2007-3956) 2009 1,945,000 ERC Advanced-EU-StemCellMark 2009 2,106,000 Netherlands Proteomics Center (Bsik Proteomics NPC II) 2009 220,000 EU/TORNADO KBBE 222720 2009 261,216 NIH/MIT Sub award 5710002735 2010 376,905 ZonMw/NGI/Pre-Seed 936.10.01 2010 400,276 Netherlands Institute for Regenerative Medicine (NIRM/Clevers) 2011 874,000 NWO/ZonMW TAS award 2011 2,000,000 VvHL /KPB/AvR/2012.010 2011 375,000 Cardio Vasculair Onderzoek Nederland (CVON 2011-12 HUSTCARE 1) 2012 468,160 HNPCC Colon adenomas biobank, (Leipzig/ 0316065A) 2012 181,072 Cancer Genomics Center (NWO Zwaartekracht)III 2013 938,115 EU-FP7 Supresstem 2013 1,122,911 Helmsley Trust-pilot 2013 119,841 UMCG/Skolkovo 2013 1,752,724 NWO/ZonMW MKMD 2013 299,670 Philips/NLY0-4520129424 2013 166,000 Stand up 2 Cancer-AACR 2014 2,817,000 Leducq 2014 471,000 Helmsley Trust-extension 2014 500,000 KWF/HUBR 2014 285,000

Patents etc. (2008-2014) PCT/NL2008/050543: A method for identifying, expanding, and removing adult stem cells and cancer stem cells (LGR5/6 as a marker for stem cells) PCT/NL2010/000017: Culture medium for epithelial stem cells and organoids comprising said stem cells PCT/IB2011/002167: Liver organoid, uses thereof and culture method for obtaining them PCT/EP2012/056950: Compounds PCT/IB2012/052950: Culture media for stem cells CT/IB2012/057497: A rapid quantitative assay to measure cftr function in a primary intestinal culture model

- 15 - Previous research

TCF factors, mediators of Wnt signaling in development and cancer In 1991, we reported the cloning of a T cell specific transcription factor that we termed TCF1. Related genes exist in genomes throughout the animal kingdom. We have shown in frogs, flies and worms that upon Wingless/Wnt signaling, ß-catenin associates with nuclear TCFs and contributes a trans- activation domain to the resulting bipartite transcription factor, and designed the widely used pTOPFLASH Wnt reporters. In the absence of Wnt signaling, we found that Tcf factors associate with proteins of the Groucho family of transcriptional repressors to repress target gene transcription.

The tumor suppressor protein APC forms the core of a cytoplasmic complex which binds ß-catenin and targets it for degradation in the proteasome. In APC-deficient colon carcinoma cells, we demonstrated that ß-catenin accumulates and is constitutively complexed with the TCF family member TCF4. In APC-positive colon carcinomas and melanomas, dominant mutations in ß-catenin render it indestructible, providing an alternative mechanism to inappropriately activate transcription of TCF target genes.

In mammals, physiological Wnt signaling is intimately involved with the biology of adult stem cells and self-renewing tissues. We were the first to link Wnt signaling with adult stem cell biology, when we showed that TCF4 gene disruption leads to the abolition of crypts of the small intestine, and that TCF1 gene knockout severely disables the stem cell compartment of the thymus. The Tcf4-driven target gene program in colorectal cancer cells is the malignant counterpart of a physiological gene program in selfrenewing crypts. Amongst the Wnt target genes, the Lgr5 gene is unique in that it marks small cycling cells at crypt bottoms. Utilizing several knock-in mouse models, we have shown that these cells represent the epithelial stem cells of the small intestine and colon. Moreover, using the same mouse models, we have described in detail the characteristics of the small intestinal crypt stem cells. They don't adhere to the commonly accepted views on adult stem cells: 1) They are not quiescent but divide once each day for the lifetime of a mouse. 2) They divide symmetrically and subsequently compete for niche space in a process called neutral competition. 3) The niche is formed by stem cell daughters: Paneth cells that secrete Wnt and EGF and carry Notch ligands. 4) The stem cell hierarchy displays plasticity in that committed daughters can be 'routinely' recruited back to become stem cells upon damage to the stem cell compartment. We have shown that Lgr5 (as a single marker) also identifies stem cells in multiple additional organs including the stomach, hair follicles, kidney, liver and pancreas. A homologue, Lgr6, marks stem cells in the skin. We showed in mice that intestinal neoplasia arise from Lgr5 stem cells and not from other cell types. Moreover, we demonstrated by lineage tracing that -in established adenomas-, rare Lgr5 stem cells act as adenoma stem cells.

The Lgr proteins constitute receptors for the secreted R-spondins, known to act as enhancers of Wnt signal strength. Lgr5 is crucial for stem cell activity: Rnf43 and Znrf3 are two tumor suppressor proteins that are encoded by Wnt target genes. They constitute transmembrane proteins that ubiquitinate and remove surafec-expressed Frizzleds, thus acting as a negative feedback-loop in the Wnt cascade. When R-spondin binds Lgr5, R-spondin can also bind these E3 ligases and remove them -together with Lgr5- from the cell surface, thus removing a negative Wnt regulator and robustly boosting Wnt signal strength.

We have previously established the crucial role of at least two other signaling pathways in intestinal epithelial biology. The Bmp pathway restricts the presence of crypts, while Wnt signaling intimately interacts with the Notch cascade to drive proliferation and inhibit differentiation in intestinal crypts and adenomas. Based on these insights, we have developed culture technologies for unrestricted expansion of single Lgr5 stem cells. This Lgr5/R-spondin-based culture system was first established for the outgrowth of single mouse or human Lgr5 stem cells into ever-expanding mini-guts It was then extended to the culture of mini-stomachs, liver-, pancreas- and prostate organoids. These epithelial organoid cultures are genetically and phenotypically extremely stable, allowing transplantation of the cultured offspring of a single stem cell from colon and from liver into multiple recipient mice. Stem cells can also be expanded from biopsies taken from patients with hereditary disorders such as Cystic Fibrosis and from a variety of cancers. As a proof-of-concept, the CFTR locus was repaired in single gut stem cells from two pediatric Cystic Fibrosis patients, using CRISPR/Cas9 technology in conjunction with homologous recombination. Repaired stem cells were clonally expanded into mini- guts and shown to contain a functional CFTR channel.

- 16 - Future research

In the next five years, we will continue to create new knock-in mouse models to study adult stem cell biology and Wnt signaling. The genes-to-be-modified are typically selected by differential gene expression analysis of sorted Lgr5 stem cells. Ongoing efforts are targeting Lgr5 stem cell-specific genes like Nav1, CDCA7, Troy, OlfM4, several Zn finger genes, Musashi2 and several uncharacterized long non-coding RNAs.

With the established mouse models based on the stem cell marker Lgr5, we have been in the unique position to characterize, isolate, analyze and genetically modify intestinal stem cells in great detail. A particularly surprising development has been the recognition that Lgr5 marks in essence marks stem cells in all internal organs. Based on this insight, we have rapidly developed 3D culture systems for human small intestine, colon, stomach, liver, pancreas, prostate, lung and mammary gland. These organoids are amenable to all standard cell-biological and molecular manipulations, including confocal imaging, mass-spec analysis of proteins, deep-sequencing of DNA and RNA, lentiviral transduction and CRISPR-mediated gene modification. Thus, most future efforts of the lab will go to exploiting the R-spondin/Lgr5-based organoid culture systems. Two main objectives will be pursued: 1) Developing human organoid technology for disease modeling, by establishing cultures from patient biopsies. Two groups of diseases will be pursued: on the one hand primary hereditary disorders of liver, gut, lung etc. and on the other hand a variety of carcinomas of the same organs. 2) Developing human organoid technology for regenerative medicine strategies, either by using healthy donor organoids or by genetically modifying mutated genes using the CRISPR technology (gene therapy in adult stem cells). Primary focus will be on regenerative strategies for the human intestine and the liver. A large NWO-TAS grant has been obtained with Prof Edward Nieuwenhuis from the Children's Hospital in Utrecht for this purpose.

Another line of research will focus on the remarkable genetic stability of Lgr5 stem cells in vivo as well as in culture. Both at the structural level and at the sequence level, the cells have a remarkable capacity to repair genetic changes. Although we are no experts on DNA repair, we hope to import this knowledge through collaborations with experts in mitotic control and DNA repair.

Societal relevance and societal impact (2008-2014)

The work on Lgr5, R-spondins and organoid technology has so far led to five patent families. These patent families have served as the basis for a non-for-profit foundation, the HUB, established by the KNAW and the UMC-U. The foundation builds extensive biobanks (owned by the UMC-U but exclusively available to the HUB) for cancer of the colon, prostate, pancreas, lung and breast. A two- year exercise with the Medical Ethical Committee of the UMC-U have allowed us to develop patient consent forms and protocols through which biobanks can now be built that are annotated in terms of whole genome sequence, coupled to clinical data. These biobanks can be made available in the context of academic collaborations (currently already supported through an NWO Zwaartekracht consortium grant and an international Stand Up 2 Cancer-grant), but they can also be made available to commercial partners. Multiple contracts have been/are being signed with pharma companies who are interested to apply the organoid technology for development of drugs for cancer and for cystic fibrosis. Moreover, the technology can be used to guide cancer drug therapy in a personalized fashion, by exposing tumor organoids in vitro to large series of drugs and determine drug resistance. We are currently validating the use of organoid technology for personalized medicine in cancer. The financial resources that are generated through these commercial interactions are reinvested in organoid research within the HUB and in the labs of HUB partners. Independent of this, the developed organoid technologies hold promise for the use Lgr5 stem cells in regenerative medicine applications, potentially replacing whole organ transplantation from cadaveric donors by cultured and frozen stem cell transplantation originating from live volunteer donors.

- 17 -

- 18 -

Menno Creyghton

Key publications

Vermunt, M.W., Reinink, P., Korving J., de Bruijn, E., Creyghton, P.M., Basak, O., Geeven, G., Toonen, P.W., Lansu, N., Meunier, C., Heesch, S., Netherlands Brain Bank; Clevers, H., de Laat, W., Cuppen, E. and Creyghton M.P.. Large scale identification of co-regulated enhancer networks in the adult human brain. Cell Rep, in press.

Creyghton, M.P., Cheng A.W., Welstead G.G., Kooistra T., Carey B.W., Steine E.J., Hanna, J., Lodato, M.A., Frampton G.M., Sharp P.A., Boyer L.A., Young, R.A., Jaenisch R. (2010) Histone H3K27ac seperates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci USA 107(50):21931-6.

Hanna, J., Saha K., Pando B., van Zon B, Lengner, C.J., Creyghton, M.P., van Oudenaarden A., Jaenisch, R. (2009) Direct reprogramming is a stochastic process amenable to acceleration. Nature 462(7273):595-601.

Creyghton, M.P., Markoulaki, S., Levine, S., Hanna, J., Lodato, M.A., Sha, K., Young, R.A., Jaenisch, R., Boyer, L.A. (2008) H2AZ if enriched at polycomb complex target genes in ES cells and is necessary for lineage commitment. Cell 135(4):649-61.

Hanna, J., Markoulaki, S., Schorderet, P., Carey, B.W., Beard, C., Wernig, M., Creyghton, M.P., Steine, E.J., Cassady, J.P., Foreman, R., Lengner, C.J., Dausman, J.A., Jaenisch, R. (2008) Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133(2):250-264.

- 19 - Dr. Menno P. Creyghton Neuro epigenetics

Group members Postdocs None Graduate students Maartje Vermunt, Sander Tan, Bas Castelijns Technicians Peter Reinink

Curriculum vitae group leader

Name Menno P. Creyghton Date of birth 30 May 1975 Nationality Dutch

Education/positions 1999 MSc Free University Amsterdam (VU) 2000-2006 PhD Netherlands Cancer Institute, Bernards lab (Amsterdam, The Netherlands) 2006-2011 Postdoc The Whitehead Institute, Jaenisch lab (Boston, USA)

2011-present Junior Group leader, Hubrecht Institute

Awards 2006-2008 Postdoctoral Fellowship from the Dutch Cancer Society

Other activities •

Thesis advisor for • Marith W. Vermunt • Sander Tan • Bas Castelijns

Invited speaker on meetings (2008-2014) • Universtity of Milano, Gene regulation and human disease, Milan Italy (2014) Large scale identification of co-regulated enhancer networks in the adult human brain. • NBB, PSYbrain Tissue Research, Amsterdam, The Netherlands (2014) Large scale identification of co-regulated enhancer networks in the adult human brain. • OGMBT annual meeting on life sciences, Vienna Austria (2014). Large scale identification of co- regulated enhancer networks in the adult human brain. • Epigenetics and Stem cells, Abcam conference, Cambridge UK (2012). Large scale epigenetic profiling of enhancers in the human brain. • CEMM Centre for Molecular Medicine, Vienna, Austria (2011). Comparative epigenomic profiling of distal enhancers during lineage commitment. • Next Generation Sequencing Meeting Benelux, Utrecht, The Netherlands (2011). Comparative epigenomic profiling of lineage commitment: Data Analysis. • MIT biology forum, Cambridge USA (2010). Comparative epigenomic profiling of distal enhancers during lineage commitment.

Grants (2008-2014) 2013-2015 Parkinsons foundation research grant € 98,000 2012-2017 Dutch Cancer Society (KWF) research grant € 550,000

- 20 - Previous research

My research has focused mainly on understanding and controlling cellular state. In particular the reprogramming of adult cells into embryonic stem cells was a major focus during my postdoctoral research in the laboratory of Rudolf Jaenisch. For instance, we showed that fully differentiated cells can be reprogrammed to embryonic stem cells (human and mouse) and used these in models for regenerative medicine. Here my interest in these processes shifted from trying to get reprogramming to work by trial and error using genetic screens based on my PhD research towards understanding the massive epigenetic changes that dictate these cell state changes. This would allow a more educated approach towards finding the master regulators of transcription that define cell state.

At the time we used ChIP-array technology to map epigenetic signatures in mouse embryonic stem cells. We discovered an interesting correlation between the histone variant H2AZ and the repressive polycomb mark at promoters that is specific for ES cells. This observation provided clues about the difference between pluripotent and more restricted cells, the first being noisier and allowing the coexistence of active and repressive states on a large scale. Around this time ChIP Sequencing was introduced in the community and we immediately adapted this technique and developed several analysis pipelines to interpret the large scale datasets emerging from these platforms. As chromatin marks at distal enhancers were found to be highly variable between cell lines we became interested in the possible role of these elements during cell fate determination. We observed that distal enhancers appear to contain much of the information that specifies the developmental state of adult tissue. Furthermore we found a unique chromatin signature that discriminates between active enhancers and those in a predetermined state. This allowed us to make predictions on developmental restrictions of tissue as well as the identity of transcription factors that are instrumental in establishing these.

As we noticed the detrimental effects of cell culture on epigenetic landscapes as well as the extremely low evolutionary conservation of enhancers between species we recently shifted our attention towards studying adult human tissues. The notion that while a cell is defined by its epigenetic profile, we essentially know very little about the epigenetic state of adult tissue in relevant its relevant context (human tissue) is perplexing. This field is entirely new and unexplored and has many unresolved challenges ahead such as dealing with individual variation and heterogeneity. We got particularly interested in studying enhancers in the brain due to its complex nature and functional diversity. Complex issues such as functional regionalization, behavior, and diseases of the brain all have strong epigenetic components at their base leaving a rich and unexplored field. We have now finalized the analysis on ChIP-Seq datasets for 141 anatomical samples in the adult human brain. In this work we identify disease associated variations that predispose to neurodegenerative disorders as altered enhancers in the brain. Furthermore we have developed a new method to deal with the heterogeneous aspects of complex tissue by using co-regulation across many anatomical sites to couple enhancers. In the process we have determined several interesting venues for follow up research which are outlined below.

Future research

Conventional studies on the epigenetic state of the genome are conducted in cell culture or tissue from laboratory animals. Both of these are epigenetically substantially different from adult human tissue. Thus very little is known about the human epigenome in its relevant context especially in complex heterogeneous tissue such as the brain. Our research currently focuses on several major focus points laid out in the emerging field of neuro epigenetics.

First we have developed computational methods to predict genome scale epigenomic information at single cell type resolution in heterogeneous cortical samples. We are currently developing RNA FISH to detect eRNAs associated with enhancers in brain sections to prove the robustness of our computational predictions and show cell type specificity for active enhancers in post mortem brain tissue. We are complementing this approach with acute post mortem isolation and FACS analysis to obtain pure populations of astrocytes and microglia through collaboration with the UMC and the Netherlands Brain Bank. We aim to assign all district enhancers identified by us in the brain to single cell types present.

- 21 - Furthermore current criteria used to determine the outcome of trans-differentiation protocols for regenerative medicine or in vitro modeling of cells from the brain often rely on morphological criteria or marker gene expression which are at best indicative of functional outcome. Thus current in vitro protocols have only yielded limited success. Based on single cell type information described above our data can for the first time define the target cell epigenomic state in vivo which will serve as a quality check with predictive power as to the transplantable success of in vitro generated material using ESCs and iPSCs. We aim to use these true in vivo epigenomic states to check neural cell types generated previously using ESCs and iPSCs. We will use these to identify factors that can aid in generating more relevant transplantable material or allow in vivo trans-differentiation without the generation of an embryonic state.

In addition we are using the resource generated to understand the late evolutionary changes that have shaped the features of the human brain that are unique to our species. We are finalizing large scale datasets in Macaque and Chimp brain and have identified several interesting state transitions at distal enhancers between these species. We are currently using the CRISPR-CAS9 system to model these changes in mouse brain in order to evaluate their effect on gross brain anatomy as well as behavior.

Finally we are using the data generated to explain the pervasive association between non coding sequence variants and several complex diseases of the brain including neurodegenerative disease and cancer. We have identified altered enhancers associated with Parkinson’s disease, Alzheimer’s disease, ALS, glioblastoma as well as several other behavioral disorders. Chromosome conformation capture is used to identify the targets of these enhancers and their activity will be monitored in mouse models at a large scale.

Societal relevance and societal impact (2008-2014)

Understanding the brain is one of the key challenges our species is faced with and requires a full understanding of the neural networks within the brain as well as the diverse cell types that specify these networks. As the genome is identical in each cell the diversity between cell types is determined solely by the epigenetic landscape that regulates the genome. Being both species specific as well as affected by environmental changes such as tissue culture stress, understanding the nature of this landscape in human tissue is the only possible venue to understand the human brain and its complex interaction with the environment.

Our research deals with a major barrier to achieving this goal being the difficulty to interpret data from heterogenic tissue. This poises us to gain unique insight into the function of the most fascinating organ in our body. Following this, the methods we employ can be translated to other complex organ systems as well.

Our datasets generated in the human brain are available as a resource for the scientific community and are expected to generate substantial follow up research venues into the complex nature of the human brain which we and other scientists can explore. We thus expect that our resource will be of immediate interest to scientists that study brain function, genetic/ environmental interactions as well as for scientists that have an interest in evolution or behavioral and neurodegenerative disorders.

The data shed light on the interaction between genome variation and emergence of complex age related diseases such as Parkinson’s disease and Alzheimer’s disease and different types of dementias which selectively affect the human species. Generating this data in relevant tissue is therefore of exceptional interest since recent studies demonstrate that promising drugs based on model organisms fail in clinical trials, likely because regulatory elements in the genome are highly species specific. The resource that we have generated is thus important to identify candidates that can be targeted for therapeutic purposes or serve as biomarkers to allow early age detection of degeneration. The latter is beneficial for treatment success especially in Alzheimer’s disease.

In this light our data has an impact on the European Union’s spearheaded fight on Alzheimer’s disease and other dementias as well as their focus on healthy aging. As more than twenty percent of Europeans will be aged 65+ years old or more by 2025 the prevalence of age related disorders such as cognitive decline and neurodegenerative disease is rising. Age related disorders of the brain are a primary factor of disability-adjusted life years worldwide especially in developed countries. This is

- 22 - accompanied by more than 30 million new cases of dementia predicted to occur each year translating into substantial increases in costs for medical care. With additional brain tissue data including patient data being added to the resource we have created the data is highly relevant for these EU initiatives. Furthermore, the data generated by us are of exceptional interest to researchers within the Human Brain Project that similarly aims to understand the interplay between genetic and environmental effects on brain function and brain disease.

Finally, comparison of in vitro generated material using directed differentiation of human induced pluripotent stem cells (iPSCs) to the true epigenetic in vivo state of neurons in an adult human brain will identify those differentiation protocols that reach the desired endpoint for regenerative medicine purposes in neurodegenerative disease and those protocols that yield cell that are epigenetically flawed. For these flawed systems we can identify missing factors in the current protocols by comparing them to the epigenomic state of healthy neurons. As such our work is already getting attention of biotech companies that attempt to harness the potential of iPSCs in regenerative medicine.

- 23 -

- 24 -

Edwin Cuppen

Key publications

Rat Genome Sequencing and Mapping Consortium (2013). From sequence to phenotype variation in the laboratory rat. Writing group: Baud A, Hermsen R, Guryev V, Gauguier D, Stridh P, Olsson T, Holmdahl R, Graham D, McBride MW, Foroud T, Fernandez-Teruel A, Hubner N, Cuppen E, Mott R, Flint J. Sequencing group: Hermsen R, Hummel O, Lansu N, Patone G, Ruzius FP, de Bruijn E, Hauser H, Atanur SS, Aitman TJ, Flicek P, Adams DJ, Keane T, Saar K, Hubner N, Guryev V, Cuppen E. Combined sequence-based and genetic mapping analysis of complex traits in outbred rats. Nat Genet 45(7):767-75.

Kettleborough RNW*, Busch-Nentwich EM*, Harvey SA*, Dooley CM, de Bruijn E, van Eeden F, Sealy I, White RJ, Herd C, Nijman IJ, Fényes F, Mehroke S, Scahill C, Gibbons R, Wali N, Carruthers S, Hall A, Yen J, Cuppen E*, Stemple DL* (2013). A systematic genome-wide analysis of zebrafish protein- coding gene function. Nature 496(7446):494-7.

Kloosterman WP, Tavakoli-Yaraki M, van Roosmalen MJ, van Binsbergen E, Renkens I, Duran K, Ballarati L, Vergult S, Giardino D, Hansson K, Ruivenkamp CA, Jager M, van Haeringen A, Ippel EF, Haaf T, Passarge E, Hochstenbach R, Menten B, Larizza L, Guryev V, Poot M, Cuppen E (2012). Constitutional chromothripsis rearrangements involve clustered double-stranded DNA breaks and nonhomologous repair mechanisms. Cell Rep 1(6):648-55.

Harakalova M, van Harssel JJ, Terhal PA, van Lieshout S, Duran K, Renkens I, Amor DJ, Wilson LC, Kirk EP, Turner CL, Shears D, Garcia-Minaur S, Lees MM, Ross A, Venselaar H, Vriend G, Takanari H, Rook MB, van der Heyden MA, Asselbergs FW, Breur HM, Swinkels ME, Scurr IJ, Smithson SF, Knoers NV, van der Smagt JJ, Nijman IJ, Kloosterman WP, van Haelst MM, van Haaften G, Cuppen E. (2012). Dominant missense mutations in ABCC9 cause Cantú syndrome. Nat Genet 44(7):793-6.

Guryev V, Saar K, Adamovic T, Verheul M, van Heesch S, Cook S, Pravenec M, Aitman T, Jacob H, Shull JD, Hubner N, and Cuppen E. (2008) Distribution and functional impact of DNA copy number variation in the rat. Nat Genet 40(5):538-45.

- 25 - Prof. Dr. Edwin Cuppen Genome Biology and Medical Genetics

Group members (August 2014)

Postdocs Joep de Ligt, Ruben van Boxtel, Ewart Kuijk, Anna van Tetering, Ies Nijman (UMCU), Terry Vrijenhoek (UMCU), Pjotr Prins (UMCU), Martin Elferink (UMCU) Graduate students Myrthe Jager, Francis Blokzijl, Roel Hermsen, Marlous Hoogstraat (UMCU) Technicians Ewart de Bruijn, Lisanne de la Fonteijne, Mark Verheul, Esther Hazendonk, Pim Toonen, Nico Lansu Bioinformaticians Sander Boymans, Robert Ernst (UMCU), Annelies Smouter (UMCU)

Curriculum vitae group leader

Name Edwin Cuppen Date of birth 11 August 1970 Nationality Dutch

Education/positions 1988 - 1994 MSc, cum laude, at the Agricultural University of Wageningen 1995 - 1998 PhD, University of Nijmegen, Faculty of Medical Sciences, Department of Cell Biology (head Prof. B. Wieringa, topic protein tyrosine phosphatases) 1999 - 2001 Postdoc Netherlands Cancer Institute Amsterdam and Hubrecht Institute Utrecht (head Prof. R. Plasterk, topic G protein-coupled receptors in C.elegans) 2002 - 2005 Junior group leader, Hubrecht Institute 2007 - 2012 Professor of Genome Biology, Utrecht University 2006 - 2009 Group leader (tenured), Hubrecht Institute 2009 - present Group leader (tenured, 50%), Hubrecht Institute 2009 - present Professor of Human Genetics, Head of Research (tenured, 50%), Department of Medical Genetics, Center for Molecular Medicine, University Medical Center Utrecht

Awards 2013 NWO Vici Award (1.5 M€) 2005 European Young Investigators (EURYI) Award (1.2 M€, European Science Foundation) 2007 Netherlands Bioinformatics Centre (NBIC) Venture Challenge (€ 30,000)

Other activities Academic advisory activities 2013 - present Member 'wetenschappelijke raad KiKa' (scientific council foundation Children Cancer Free) 2010 - present Advisory Board Liverpool Centre for Genomic Research 2010 - present Advisory Board Knockout Rat Consortium (www.knockoutrat.org) 2007 - 2008 Member Scientific Advisory Board ELSYS (Young European Scientists in Life Science 2008 Member scientific site visit committee CGM, Paris, France 2005 Member scientific site visit committee Roslin Institute, Edinburg, UK

Commercial advisory activities (remunerated) 2014 - present Scientific Advisor BlueBee (hardware acceleration) 2013 - present Scientific Advisor Elly Lilly (rat genomics) 2012 - present Scientific Advisory Board Cergentis BV (targeted sequencing technology) 2012 - present Scientific Advisory Board Genalice (high volume data analysis) 2008 - 2012 Scientific Advisor Philips Research (DNA analysis technology) 2006 - present Scientific Advisory Board InteRNA Technologies BV (miRNA-based therapeutics)

Other national and international scientific activities 2013 Scientific committe 2nd International Conference on Genomics, Ghent, Belgium 2013 Chair program committee Federa Dag Next-generation sequencing 2013 2007 - present Organizer annual Cold Spring Harbor/Wellcome Trust meeting ‘Rat genomics and models’

- 26 - 2010 - present Steering Committee member Center for Personalized Cancer Treatment (www.CPCT.nl) 2010 - present Steering Committee member EU FP7 program EURATRANS (www.euratrans.eu) 2012 - present Mentor Steyn Parve coaching program UMC Utrecht 2011 - 2013 Director Netherlands Center for Genome Diagnostics (CGD) 2008 - 2012 Organizer annual Dutch Next-Generation Sequencing Users Meeting 2009 - 2012 Chair Jury X-track Honors Program 2010 Co-organizer first European (ESF) next-generation sequencing meeting 2007 - 2009 Member program committee NWO Computational Life Sciences II.

Thesis advisor for promotion date • Harma Feitsma 04-06-2008 • Karin Brouwer* 06-03-2009 • Judith Paridaen* 07-05-2009 • Josien van Wolfswinkel* 16-12-2009 • Kirsten Sporendonk* 17-12-2009 • Sam Linsen 08-02-2010 • Joram Mul 30-06-2010 • Ruben van Boxtel 23-08-2010 • Michal Mokry 23-11-2011 • Anne Lagendijk* 13-12-2011 • Jos Poell 16-01-2012 • Meltem Isik* 22-05-2012 • Turan Demircan* 11-09-2013 • Magdalena Harakalova 07-11-2013 • Paul Essers* 05-12-2013 • Floor Twis* 16-01-2014 • Robert Magliozzi* 21-01-2014 • Dimphna Meijer* 06-02-2014 • Daniil Simanov* 15-04- 2014 • Jessica van Setten* 26-06-2014 • Sebastiaan van Heesch 02-07-2014 • Marlous Hoogstraat 30-10-2014 • Margarita Zacharogianni* scheduled 20-11-2014 • Jihoon Kim* scheduled 13-01-2015 • Roel Hermsen scheduled 21-01-2015 *not direct thesis supervisor, only promoter

Invited speaker on meetings (2008-2014) 01-06-2014 ESHG, Milan, Italy 21-03-2014 Master class Stem Cells in Cancer, Maastricht 10-02-2014 Keynote Netherlands Proteomics Center, Utrecht 02-11-2013 Alumni dag 25 jaar, Wageningen 18-09-2013 MRC-CSC seminar series, London, UK 21-06-2013 CRG seminar series, Barcelona, Spain 27-05-2013 CPCT scientific symposium, Utrecht 13-09-2013 Achmea Invitional conference, Amsterdam 06-03-2013 SmartMix symposium, Nijmegen 25-01-2013 Seminar series Netherlands Proteomics Centre, Utrecht 20-09-2012 NVHG najaarssymposium, Papendal 17-09-2012 Keynote, German Society for Research on DNA Repair conference, Munich, Germany 17-02-2012 Advances in Genome Biology and Technology (AGBT), Marco Island, Florida, USA 10-08-2012 Studium General, Utrecht 18-09-2012 Keynote Flemish Training Network Life Sciences meeting, Leuven, Belgium 10-02-2012 Symposium Moleculaire diagnostiek, Utrecht 19-12-2012 NVVI international meeting, Noordwijkerhout 28-08-2012 Keynote UK NGS meeting, Nottingham, UK 09-11-2012 ASHG, San Francisco, USA 12-11-2012 Keynote Genetics & medicine symposium, Louisville, USA

- 27 - 06-12-2012 CSHL/WT Rat Genomics & Models. Hinxton, UK 09-06-2011 Copenhagenomics, Kopenhagen, Denmark 03-10-2011 Minisymposium 'Genome Evolution and Genome Stability', Leuven, Belgium 25-01-2011 Café Scientifque, Amsterdam 11-03-2011 Seminar Series, Kuala Lumpur, Maleisië 10-03-2011 AITBIOTECH & BRETSS Seminar, Singapore 24-10-2011 Cancer center seminars, Utrecht 07-06-2011 Laboratory Animals Science seminar, Hannover, Germany 12-05-2011 Science Café, Wageningen 26-04-2011 Keynote Next Generation Sequencing Congress, Boston, USA 10-09-2010 WT meeting Signaling to chromatin, Hinxton, UK 09-11-2010 NCRI meeting Liverpool, UK 20-05-2010 Annual HUGO meeting. Montpellier, France 09-03-2010 SOLiD users meeting, Antwerpen, Belgi, SOLiD users meeting. 29-01-2010 NXTGNT kickoff meeting, Gent, Belgium 25-11-2010 Nederlands Netwerk Onderzoek Farmacogenetica (NNOF) Meeting, Utrecht 30-08-2010 ESF international NGS meeting, Leiden 19-11-2010 Keynote Najaarssymposium NVHG, Papendal 02-04-2010 Public lecture Van Gogh museum, Amsterdam 08-11-2010 Seminar series University of Liverpool, Liverpool, UK 15-09-2010 Keynote 56th Brazilian Congress of Genetics, Guaruya, Brazil 01-12-2010 The 18th International Workshop on Genetic Systems in the Rat, Kyoto, Japan 18-10-2010 ABI/IC/Canceropole IdF- NGS Symposium, Paris, France 15-12-2009 Norway next-generation sequencing users meeting. Oslo, Norway, 05-02-2009 Advances in Genome Biology and Technology (AGBT), Marco Island, USA 10-03-2009 SOLiD Roadshow, Dresden, Germany 27-02-2009 SOLiD Roadshow, Milan, Italy 08-06-2009 INSERM symposium, Nantes, France 17-11-2009 Agilent Roadshow, Gent, Belgium 07-07-2009 Second Belgium-Netherlands-Luxembourg Next-generation sequencing users meeting, Utrecht 13-10-2009 MIP-Tec, Basel, Switzerland 20-10-2009 NGS Symposium, Paris, France 29-09-2009 LUMC seminar series, Leiden 28-06-2009 Keynote ISMB SIG, Stockholm, Sweden 10-06-2009 SOLiD users meeting, Milan, Italy 01-10-2009 1st Systems Genetics Symposium, Groningen 08-05-2009 Keynote NXTGNT, Gent, Belgium 22-03-2009 Keynote Advances in targeted therapies, Mandelieu, France 16-10-2009 Afscheidsymposium Pim Zabel, WUR, Wageningen, 29-10-2009 NCMLS Masters course Radboud University, Nijmegen 30-10-2009 Seminar series University of Lausanne, Lausanne, Switzerland 08-10-2009 MDC Summer Meeting. Berlin, Germany 2008, no data available anymore

Grants (2008-2014) • NWO Vici (€ 1,500,000 2014-2019): Dissecting structural variation • KWF Stand up to cancer (€ 400,000 2013-2017): Cancer organoid genomics • NWO Zwaartekracht (€ 500,000 2013-2022): Cancer Genomics Netherlands • NGI Zenith (€ 100,000 2012-2016): Genetic integrity of adult stem cells • Center for Genome Diagnostics (CGD), Netherlands Genomics Initiative (coordinator, € 1,000,000 2011-2013): Next-generation sequencing in diagnostics • Center for Personalized Cancer Treatment (CPCT), KWF/Alpe d’huzes (€ 500,000 2011) • NWO TOP-CW (€ 700,000 2009 – current): Rat genomics • EU FP7 EURATRANS (€ 700,000 2010-current): Rat systems genetics • EU FP7 ZFHEALTH (€ 400,000 2010-current): Zebrafish genetics • NBIC (NGI) BioRange-2 and BioAssist (€ 400,000 2010 – 2013): Next-generation sequencing data analysis • CGC-2 (NGI) (€ 500,000 2009-2013): Cancer genetics • Leducq transatlantic network (€ 100,000 2009): miRNA in heart

- 28 - • SMARTMIX (EZ and OCW) consortium (€ 1,500,000 2008 – current): Zebrafish knockout models • NCSB (NGI) (€ 200,000 2008 – 2013): Cancer systems genetics • NBIC (NGI) Biorange-1 (€ 100,000 2006-2009): Bioinformatics analysis for mutation analysis • EU FP6 ZFMODELS (€ 700,000 2004-2009): Zebrafish knockout models

Patents etc. (2008-2014) • Weijzen, S., Schaapveld, R., Bourajjaj, M., van Haastert, R., Griffioen, A., van Beijnum, J., Cuppen, E., Berezikov, E., van Puijenbroek, A., Gommans, W., Negar, B., (2011). Mir-190b for treating neo-angiogenesis. US application no 61/521,917 and 61/522,346 • Weijzen, S., Schaapveld, R., Bourajjaj, M., van Haastert, R., Griffioen, A., van Beijnum, J., Cuppen, E., Berezikov, E., van Puijenbroek, A., Gommans, W., van Noort, P., Negar, B., (2011). Mir-142 for treating neo-angiogenesis. US application no 61/521,931 • Weijzen, S., Schaapveld, R., Bouraijaj, M., van Haastert, R., Griffioen, A., van Beijnum, J., Cuppen, E., Berezikov, E., van Puijenbroek, A., Gommans, W. (2011). Mir for treating neo- angiogenesis. EPC:11150645.7-1212

Previous research

After my PhD in cell biology and signal transduction, I specialized in genomics and genetics. While my early work focused on genomics studies in model organism, mainly involving rats and zebrafish, over time my scientific interests developed towards functional and personal genomics. I have always combined interestes in technology development with studies on biological problems in the area of genome biology and function and as a consequence have been able to capitalize on developments in DNA-based genomics technologies in a very early stage. For example, my lab was one of the first to generate gene knockout models in the rat using target-selected mutagenesis, which has resulted in a range of technological papers as well as several dozen papers on the biological characterization of the resulting rat knockout models. These rat models (e.g. Mc4r, Sert, Tp53) are now widely used in biomedical research and are publically available through common commercial suppliers. Furthermore, by generating and exploiting rat genomics data my lab was also one of the first to demonstrated the widespread effects of copy number variation on gene expression levels. As recognition of these efforts, I was awarded a prestigious European Young Investigators Award in 2005 for my work on naturally occurring and induced genetic variation in the laboratory rat.

In my current work, I combine experimental methods, including next-generation DNA sequencing technology and animal model studies, with bioinformatics approaches to identify functional elements in genomes and to understand the effects of genetic variations under normal and disease conditions. My group has specialized in the detection of structural variation and was the first to show that a novel phenomenon called chromothripsis (massive chromosome shattering) contributes to complex germline variation and congenital disease. More recently, we also demonstrated using highly sensitive techniques that in contrast to previous reports, this mechanism is very common in both primary and metastatic tumors, at least in colorectal cancer and may therefore also be an important driver in tumorigenesis.

My research group has a long track record in high-throughput DNA analysis and was the first in Europe to implement Life Technologies NGS technology SOLiD and, most recently, the IonTorrent Personal Genome Machine. I have established close collaborations with various technology providers and published a long list of 'technology papers' in high impact journals. I have also established an extensive local, national, and international collaborative network that has resulted in joint publications and funding acquirement. Currently, we are operating the largest next-generation DNA sequencing facility of the Netherlands, including 3 SOLiD 5500XL, 2 SOLiD WildFire, 1 Illumina Hiseq2500, 2 Illumina NextSeq500, 1 Illumina Miseq, 1 IonTorrent PGM and 1 IonTorrent Proton machines. Equipment is used for diagnostic purposes in an ISO19187 certified environment (led bij J.K. Ploos van Amstel) and is also available for a very broad range of research purposes to the Utrecht Life Sciences research community through ngs.hubrecht.eu.

My own research occurs at both the Department of Medical Genetics of UMC Utrecht (human and cancer genetics) and the affiliated Hubrecht Institute (fundamental genomics in model organisms), providing a unique internationally competitive environment and allowing for clinical validation of developed technologies, as well as in-depth analysis and experimental validation of biological

- 29 - principles. Within UMC Utrecht I have initiated several translational programs for bringing NGS application into diagnostics to efficiently address a wide range of fundamental and clinically relevant questions. I am the coordinator or steering committee member of various consortia, including the EU- funded FP7 Integrated Project EURATRANS, director of the Netherlands Consortium for Genome Diagnostics and cofounder of the Centre for Personalized Cancer Treatment (collaboration between the Netherlands Cancer Insitute Amsterdam, Erasmus Medical Center Rotterdam and UMC Utrecht).

I would characterize myself as a creative scientist that balances between technology and biology in one dimension and between fundamental and patient-driven research in the other. This has resulted in a unique and highly productive situation as witnessed by my scientific and societal output. Most recently, I have been granted a prestigious NWO Vici award for a project to study causes and consequences of complex genomic structural variation. Within this project, I will use state-of-the-art techniques to molecularly and genetically characterize individual cells undergoing chromosomal missegregations to understand mechanisms underlying de novo structural variation, including chromothripsis.

Future research

My future research at the Hubrecht Insitute and UMC Utrecht will have four main directions.

1) Integrative biology. We will apply and integrate a range of genomics technologies (RNA-seq, ChIP-seq, HiC, quantitative proteomics, metabolomics, etc) to systematically dissect and understand the effects of genetic variation on molecular phenotypes. Whole genome sequencing information will form the basis for such experiments and molecular characterisations and experimental perturbations will be performed in close collaboration with other experts. The main challenge in this area is to systematically connect the different information layers, each having its own characteristics, sensitivity and (in)completeness. Furthermore, bioinformatic tools for large scale integrative omics data analyses are largely lacking. Currently, we are applying these approaches to a) inbred rats to study effects of natural occuring variation with a special focus on structural variation (in collaboration with Norbert Hubner, MDC Berlin), b) cancer cell lines and organoids to understand drug response and resistance (in collaboration with Rene Bernards and Lodewyk Wessels, NKI Amsterdam; Albert Heck, UU; Hans Clevers, Hubrecht Institute; Hans Bos, UMCU), and c) patients with congenital phenotypes to dissect consequences of de novo SVs (in collaboration with Wigard Kloosterman and Paul Coffer, UMCU; Jeroen Bakkers, Hubrecht Institute).

2) Cause and consequences of chromothripsis. Genomic mutations and chromosomal rearrangements drive a wide range of diseases, including congenital disorders and cancer. Complex genomic structural variations (SVs), including recently discovered chromothripsis events that scramble large genomic regions, contribute to this landscape by reshuffling and disturbing both coding and regulatory elements. Currently, there are two main outstanding questions: What are the pathogenic mechanisms resulting from such rearrangements and what triggers these potentially highly pathogenic events? I will address the first question by applying the integrative biology approach indicated above in a family trio-based setup to a range of chromothripsis patients. Preliminary work unexpectedly identified chromosomal alterations and activated mechanisms that were also observed in cancer. I will specifically dissect the molecular mechanisms by which recurrent SVs both drive congenital disorders and contribute to tumorigenesis using patient cell-based, zebrafish, and mouse models.

To address the second question and understand how complex structural arrangements arise, I will build on the observation that chromosome missegregation during cell division can result in aneuploidies and structural rearrangements. I will construct an optogenetic system to allow labelling, tracking and sorting of cultured cells with missegregating or damaged chromosomes. Individual and pooled cells will be collected at various time-points and cellular responses will be characterized at both the genomic and transcriptomic levels using NGS-based techniques. Molecular mechanisms will be unravelled by the functional manipulation of DNA-damage response systems and perturbation of environmental conditions.

3) Genomic (in) stability in adult stem cells. Adult stem cells persist throughout life to maintain tissue homeostasis and direct repair. However, they are also continuously exposed to external threads that may affect their genetic integrity, which may result in undersired pathogenic effects when

- 30 - remained undetected or unrepaired. We make use of the adult stem cell culturing technology that was recently developed in the Clevers group to study the mutational processes in various types of adult stem cells using whole genome sequencing, both in vivo and during long term in vitro expansion. Our results show that a) adult stem cells are genetically highly stable in vivo with a mutation frequency similar to germline and absence of structural variation, b) mutation frequencies increase about 20-fold upon culturing, most likely due to increased stress conditions, c) expressed coding regions are depleted for mutations, which may reflect active repair mechanisms involved, d) cycling (intestinal) AS cells display a different mutation specturm as compared to noncycling (liver) AS cells, e) spectra and frequencies differ significantly between mice and humans, and f) mutational loads in individual AS cells increase linearly with age in humans (donors between 2 and 74 years were studied). We will further build on these observations by studying more AS cell sources from the same individual (post mortem), but also by studying other model organisms to understand the role of DNA repair mechanisms and genetic integrity of AS cells in aging and tumorigenesis processes (including DNA repair and aging mutant mouse models in collaboration with Jan Hoeijmakers, and different rodents (e.g. naked mole rat) and primates). Furthermore, we will inactivate relevant DNA repair components in cultured AS cells (organoids) using the Crispr/CAS9 system to assess their involvement in DNA integrety maintenance.

4) Personalized Cancer Treatment. The CPCT is a national collaboration of academic and STZ hospitals which aims to make personalized cancer treatment available in routine patient care based on genetic analyses of tumor and metastases biopsies (member of executive committee together with Emile Voest, NKI Amsterdam and Stefan Sleijfer, Erasmus Medical Center Rotterdam). Currently, both retrospective and prospective studies are ongoing. The goal of the retrospective analyses are to identify biomarkers and/or pathways that explain targeted treatment efficiancy or resistance. Such markers will eventuelly be used in a prospective way to stratify patients towards existing or experimental treatment. My role in this initiave is to facilitate and develop targeted, whole genome and RNA sequencing assays and to integrate clinical and genetic data. At this moment, we are exploring the possibilities to establish a national medical sequencing center, which should drive NGS towards routine clinical use and reimbursement by insurance companies.

Societal relevance and societal impact (2008-2014)

The societal relevance and societal impact of my work can be assessed in different ways. First, I am an active participant in public debates around genetic and genome analyses, both involving scientists and lay man, and I am actively stimulating genetic literacy. I am regularly consulted for expert feedback in national newspapers and radio, and I have participated in a broad range of public debates, cultural festivals and 'genomics pop-up stores'. Furthermore, I have been a participant in various strategic discussions with funding bodies (NWO, KWF), government and health care insurance companies. Finally, I am scientific advisor for a range of highly innovative Dutch start-up companies in genomics and bioinformatics.

Secondly, my lab has developed techniques, assays and bioinformatic tools that are now routinely used in clinical care in the UMCU. For example, the Department of Pathology last fall replaced about 80% of its molecular tests by a customized NGS-based test in combination with bioinformatic tools developed in our lab for routine tumor mutation testing in genes like KRAS, TP53, but also amplifications in HER2, EGFR and MYC. The Department of Medical Genetics has implemented a range of targeted and whole exome sequencing approaches and most recently, people in my lab (both wet lab and bioinformatics) had a key role in the implementation of the non-invasive prenatal trisomy testing in Utrecht. Currently, I am one of the drivers behind the establishment of a national medical sequencing center, which should bring NGS-based approaches in a safe and timely manner to the patient and society.

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Eelco de Koning

Key publications (2008-2014)

Ellenbroek JH, Tons HA, Westerouen van Meeteren MJ, de GN, Hanegraaf MA, Rabelink TJ, Carlotti F, de Koning EJ. (2013) Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice. Diabetologia; 56(9):1980-1986.

Huch M, Bonfanti P, Boj SF, Sato T, Loomans CJ, van de Wetering M, Sojoodi M, Li VS, Schuijers J, Gracanin A, Ringnalda F, Begthel H, Hamer K, Mulder J, van Es JH, de Koning E, Vries RG, Heimberg H, Clevers H. (2013) Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J; 32(20):2708-2721.

Moran A, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R, Greenbaum CJ, Herold KC, Marks JB, Raskin P, Sanda S, Schatz D, Wherrett DK, Wilson DM, Krischer JP, Skyler JS; Type 1 Diabetes TrialNet Canakinumab Study Group, Pickersgill L, de Koning E, Ziegler AG, Böehm B, Badenhoop K, Schloot N, Bak JF, Pozzilli P, Mauricio D, Donath MY, Castaño L, Wägner A, Lervang HH, Perrild H, Mandrup-Poulsen T; AIDA Study Group. (2013) Interleukin-1 antagonism in type 1 diabetes of recent onset: two multicentre, randomised, double-blind, placebo-controlled trials. Lancet; 381(9881):1905-1915.

Spijker HS, Ravelli RB, Mommaas-Kienhuis AM, van Apeldoorn AA, Engelse MA, Zaldumbide A, Bonner-Weir S, Rabelink TJ, Hoeben RC, Clevers H, Mummery CL, Carlotti F, de Koning EJ. (2013) Conversion of mature human beta-cells into glucagon-producing alpha-cells. Diabetes; 62(7):2471- 2480.

Petersen N, Reimann F, Bartfeld S, Farin HF, Ringnalda FC, Vries RG, van den Brink S, Clevers H, Gribble FM, de Koning EJ. (2014) Generation of L cells in mouse and human small intestine organoids. Diabetes; 63(2):410-420.

- 33 - Prof. Dr. Eelco de Koning Diabetes and islet neogenesis

Group members

Postdocs: Gitanjali Dharmadhakiri Graduate students: Nerys Williams Technicians: Leon van Gurp, Erik Jansen

Curriculum vitae group leader

Name: Eelco de Koning Date of birth: 5 October 1963 Nationality: Dutch

Education/positions 1983-1991 Medicine, University of Utrecht, the Netherlands 1991 M.D., University of Utrecht 1991-1994 Research fellow Diabetes Research Laboratories and Laboratory for Human Endocrinology, University of Oxford, U.K. Visiting research fellow Department of Clinical Biochemistry, University of Cambridge, U.K. and Department of Medical Cell Biology, University of Uppsala, Sweden 1994 Ph.D. Islet amyloid and islet amyloid polypeptide (supervisor prof.dr. D.W. Erkelens), University of Utrecht 1994-1999 Resident in Internal Medicine, St. Antonius Hospital, Nieuwegein, and University Medical Center Utrecht, the Netherlands 2000-2001 Fellow in Endocrinology, University Medical Center Utrecht 2001-2004 Assistant Professor, Dept. of Medicine, University Medical Center Utrecht 2004-2011 Associate Professor, Dept. of Nephrology and Dept. of Endocrinology, Leiden University Medical Center, the Netherlands 2004–2005 Visiting Associate Professor, Harvard Medical School, Joslin Diabetes Center, Dept. of Islet Transplantation and Cell Biology, Boston, U.S.A. 2006- Director Clinical Islet Transplantation Program, Leiden University Medical Center 2010- Group leader, Hubrecht Institute, Utrecht, Netherlands 2011- Professor of Diabetology, University of Leiden

Memberships • American Diabetes Association (ADA) • Endocrine Society • Dutch Royal Society of Medicine (KNMG) • Netherlands Association of Internal Medicine (NIV) • Dutch Endocrine Society (NVE) • Dutch Association for Diabetes Research (NVDO) • Dutch Transplantation Society (NTV) • International Society for Stem Cell Research (ISSCR) • European Society for Organ Transplantation (ESOT) • The Transplantation Society (TTS)

Awards 2004-2005 Mary K. Iacocca Senior Visiting Research Fellowship - Joslin Diabetes Center, Harvard Medical School, Boston 2005-2009 Career Development Award - Dutch Diabetes Research Foundation (DFN) 2003.01.008 1995 Dr. F. Gerritzen Award - Dutch Association for Diabetes Research (NVDO) 1992-1994 MRC Training Fellowship - Medical Research Council, London, U.K. 1991-1992 Bursary University of Oxford Medical School Research Fund, Oxford, U.K. 1982-1983 Bursary - Netherlands American Commission for Educational Exchange (NACEE, now Fulbright Centre), Amsterdam, the Netherlands

- 34 - Other activities 2013 President-elect European Pancreas and Islet Transplantation Association (EPITA) 2012 Local Organising Committee NICE-EPITA Islet Isolation Workshop, Leiden 2011 Board European Pancreas and Islet Transplantation Association (EPITA) 2011 Scientific Program Committee European Association for the Study of Diabetes (EASD) 2011, Lisbon 2009 Scientific Advisory Board Foundation Diabetes Research Netherlands (Stichting DON) 2006-2012 Scientific Advisory Board Dutch Diabetes Research Foundation (DFN) 2002-2009 Board Dutch Association for Diabetes Research (NVDO) 2008-2009 Co-chairman

Thesis advisor for • Rianne Ellenbroek, LUMC (2009-2013) • Siebe Spijker, LUMC (2010-2013) • Jeetindra Balak, LUMC (2011-2014) • Mathias Roost, LUMC (2011-2014) • Maaike Roefs, LUMC (2011-2014) • Jason Doppenberg, LUMC (2011-2014) • Nerys Williams, Hubrecht Institute (2011-2014)

Invited speaker on meetings (2008-2014) 2008 Waddensymposium 2009 Hesperis course, Leuven 2009 ET meeting 2010 ET Winter Meeting 2011 Oxford Endocrinology Meeting 2011 European Congress of Endocrinology, Rotterdam 2011 Workshop Regenerative Medicine, Leuven 2012 ET Winter Meeting 2012 University of Miami (invited lecture) 2012 Islet transplantation meeting Dresden 2013 Paris, DCD meeting 2013 University of Oxford, OCDEM (invited lecture) 2013 ISPAD, Gothenburg (only international meetings/lectures mentioned)

Grants (2008-2014) 2014 Co-PI Strategic Research Agreement “Pancreatic cell plasticity for the (re)generation of beta- cells in diabetes” - Juvenile Diabetes Research Foundation (JDRF) 3-SRA-2014-318-Q-R $ 400,000 2009- Scientific Director of the Diabetes Cell replacement Therapy Initiative (DCTI) consortium - Fonds Economische Structuurversterking (FES) € 7,000,000 2008- Project leader An implantable islet cell replacement device for controlled insulin release in diabetes - Dutch Diabetes Research Foundation 2008.50.001 € 1,000,000 2008- Project leader Exploration of human duct cells for beta-cell replacement therapy – Dutch Diabetes Research Foundation 2008.50.002 € 500,000

Patents etc. (2008-2014) 2010 Scaffold and a method of preparing beta cell scaffold for treatment of diabetes (patent application - WO2010/050517)

- 35 - Previous research

Since there are major differences in rodent and human beta cell and islet progenitor cell biology, studies on human cells are needed. Therefore, after having set up an islet isolation and transplantation program at the Leiden University Medical Center (LUMC), which is only one out of approximately 10 active human islet isolation transplantation centers in Europe, a research group at the Hubrecht Institute was started, merely supported by external financial sources and the LUMC, focusing on islets and islet progenitors in human pancreatic tissue. The tissue from the LUMC that we use is unsuitable for transplantation and a valuable rare source of material.

The main focus of our research is on the expansion of human pancreatic tissue and the identification, isolation and characterization of progenitor cells in the human pancreas. We have succeeded to expand human pancreatic tissue for several months in an adapted 3D culture system which was developed in the Clevers group at the Hubrecht Institute. We can clonally expand a subpopulation of human pancreatic cells that have progenitor cell characteristics. After transplantation of these expanded cells in immunodeficient mice endocrine cells are generated and human insulin can be detected in the circulation of the mice. This cumulative work over the past 4 years is now under review for publication. At the same time we have collaborated with the Clevers group on their work of LGR5+ progenitors in the murine pancreas (Huch et al, EMBO J 2013).

Active collaboration has also been set up with the van Rheenen group at the Hubrecht Institute as we developed a novel intravital imaging technique for cells transplanted under the kidney capsule (Ritsma L, Sci Transl Med 2013). This is going to be an important tool to follow labelled human progenitor cells in an in vivo environment.

After starting our group we also focused on enteroendocrine cells in the gut (L-cells) that are of major importance for postprandial insulin secretion and glucose metabolism. In collaboration with the group of Fiona Gribble in Cambridge, we showed that we can modulate the generation of these cells in vitro using our 3D culture system (Petersen N, Diabetes 2014). We have also shown that we can improve glucose metabolism in mice by modulation of the number of L-cells by notch inhibition. This new concept is now under review for publication. Despite these interesting results, this line of research has been terminated in order to focus on islet progenitor cell research.

Future research

The main focus will remain the identification and characterization of human pancreatic cells that have the capacity to differentiate into an endocrine phenotype.

More recently we established collaboration with the van Oudenaarden group at the Hubrecht Institute focusing on single cell transcriptome analysis of human pancreatic tissue, both fresh (human islets and islet-depleted tissue) and cultured human pancreatic tissue in our 3D system. The results of these experiments will guide us towards identification and characterization of interesting human pancreatic (progenitor) cell populations. Using interesting surface markers that will come out of these single cell transcriptome studies, we can zoom in on the pancreatic cells and be able to even more precisely identify and culture the cells with progenitor cell characteristics.

Recently we have been able to also culture human embryonic pancreas in a 3D culture system in collaboration with the group of Christine Mummery at the LUMC. Since both human adult and human embryonic pancreatic tissue have a typical tip-trunk expansion configuration in our 3D culture system, we will be able to perform comparative analyses on growth and differentiation patterns. We hypothesize that these comparative analysis using both in vitro and in vivo will guide us towards a better understanding of the regenerative potential of human pancreatic cells and, at the same time, will provide us with potentially relevant information about human pancreatic development.

- 36 - Societal relevance and societal impact (2008-2014)

Diabetes mellitus is a disease of endemic proportions with an estimated 350 million people suffering worldwide from the disease. It is estimated that about 10% of health care costs are attributable to diabetes in the US. The insulin-producing beta cell plays a key role in the development and progression of the disease in all types of diabetes.

In type 1 diabetes (NL 100,000 patients), which accounts for about 10% of the total number of people with diabetes, there is a rapid autoimmune destruction of the insulin producing beta cells. This disease often presents itself in children and adolescents. Insulin injections are necessary to survive but they are merely a symptomatic treatment. There is an enormous negative impact on quality of life by the burden of insulin therapy and insulin therapy cannot prevent long term diabetic complications (increased risk for amputations, blindness, cardiovascular disease and dialysis) as optimal glucose control is never achieved. Replacement of the insulin-producing cells is the only treatment that provides a cure. Postmortal pancreas and islet transplantation are 2 options of beta cell replacement therapy and these procedures are performed at the Leiden University Medical Center in the Netherlands. However, since there is a severe shortage of organ donors, this treatment is only available for a very limited number of patients with type 1 diabetes. Thus, for future therapy there is no other option than to generate insulin-producing cells from alternative cell sources in order to have an unlimited supply of insulin-producing beta cells.

Over the past years we have made an effort to reach out with our diabetes research to the general public and philanthropic organisations (national newspaper, national television, non-scientific periodicals).

The Hubrecht Institute is at the forefront of developmental and stem cell biology. The close collaboration with the human islet isolation and transplantation program in the LUMC puts the Hubrecht Institute at a unique position to focus on relevant human cells for regenerative medicine purposes and translate future findings into novel cellular therapies for patients with diabetes.

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- 38 -

Wouter de Laat

Key publications (2008-2014) de Vree PJP, de Wit E, Yilmaz M, van de Heijning M, Klous P, Verstegen MJAM, Wan Y, Teunissen H, Krijger PHL, Geeven G, Eijk PP, Sie D, Ylstra B, Hulsman LOM, van Dooren MF, van Zutven LJCM, van den Ouweland A, Verbeek S, van Dijk KW, Cornelissen M, Das AT, Berkhout B, Sikkema- Raddatz B, van den Berg E, van der Vlies P, Weening D, den Dunnen JT, Matusiak M, Lamkanfi M, Ligtenberg MJL, ter Brugge P, Jonkers J, Foekens JA, Martens JW, van der Luijt R, Ploos van Amstel HK, van Min M, Splinter E, de Laat W (2014). Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nature Biotechnol. 2014 Aug 17 Epub de Laat W, Duboule D (2013). Topology of mammalian developmental enhancers and their regulatory landscapes. Nature 502(7472):499-506. de Wit E, Bouwman BA, Zhu Y, Klous P, Splinter E, Verstegen MJ, Krijger PH, Festuccia N, Nora EP, Welling M, Heard E, Geijsen N, Poot RA, Chambers I, de Laat W. (2013) The pluripotent genome in three dimensions is shaped around pluripotency factors. Nature. 501(7466):227-31. van de Werken H, Landan G, Holwerda S, Hoichman M, Klous P, Chachik R, Splinter E, Valdes Quezada C, Öz Y, Bouwman B, Verstegen M, de Wit E, Tanay A, de Laat W. (2012). Robust 4C-seq data analysis to screen for regulatory DNA interactions. Nat Methods, 9:969-72.

Noordermeer D, de Wit E, Klous P, van de Werken H, Simonis M, Lopez-Jones M, Eussen B, de Klein A, Singer RH, de Laat W. (2011). Variegated gene expression caused by cell-specific long-range DNA interactions. Nat Cell Biol 13:944-51.

- 39 - Prof. Dr. Wouter de Laat Biomedical Genomics

Group members Postdocs: Elzo de Wit, Peter Krijger, Patrick Wijchers, Adrien Melquiond, Geert Geeven, Annette Denker, Christian Valdes Graduate students: Britta Bouwman, Erica Vos, Carlo Vermeulen (Yuva Oz, Yun Zhu and Paula de Vree writing thesis) Technicians: Marjon Verstegen, Hans Teunissen, Mark Janssen

Curriculum vitae group leader

Name: Wouter de Laat Date of birth: 14 February 1970 Nationality: Dutch

Education/positions 1988 - 1993 MSc, Biology, Utrecht University 1993 - 1998 PhD, Dept. of Genetics, Erasmus University, Rotterdam, Prof. J. Hoeijmakers, Prof. D. Bootsma, thesis: ‘Incision coordination in Nucleotide Excision Repair’ 1998 - 2000 Postdoc, Dept. of Cell Biology, Erasmus MC, Rotterdam, Prof. F. Grosveld 2000 - 2004 Staff Scientist (tenured term) Dept. Cell Biology, Erasmus MC, Rotterdam 2004 - 2008 Assistant Professor (tenured term), Dept. of Cell Biology, Erasmus MC, Rotterdam 2009 - present Professor in Biomedical Genomics, UMC Utrecht. 2008 - present Hubrecht Institute

Memberships 2009 Elected EMBO member 2014 Scientific board member KWF (Dutch Cancer Society) 2014 Supervisory Board HUB (http://hub4organoids.nl/) 2008 Editorial board member Epigenetics and Chromatin (BioMed Central) 2009 Member Collaborative KWF grant on Breast Cancer (with ErasmusMC) 2010 WP leader EU FP7 Large scale Integrating Project ‘MODHEP’ (MODelling HEPatocellular carcinoma) 2010 Member collaborative project on Nanonext NL (with ErasmusMC and Philips)

Awards 2000 NWO Vernieuwingsimpuls (VIDI), career grant (€ 650,000 for five years, PI) 2008 ERC Starting Grant (€ 1,225,000 for 5 years, PI) 2013 VICI laureate, career grant NWO (€ 1,500,000 for 5 year), Innovational Research Incentives Scheme

Other activities • Founder of Cergentis (founded July 2012): www.cergentis.com • Consultant for Cergentis (1 day/week for past 2 years, estimated 1 day/month for coming year) Within the Hubrecht Institute: • Organizer CGDB seminar series (2010-2012) • Examiner MSc rotation students (since 2014) • Introduced and helped implementing new database for the Hubrecht animal facility (AMS) Meeting organizer: • 3rd InteGeR workshop on ‘Nuclear Structure and Gene Regulation’ (Nov 9th, 2011, Utrecht; 120 participants) • Organizer and chair session ‘long-range gene regulation’ EMBO Meeting, Barcelona 2010 Scientific Book editor: • Methods (Elsevier): issue on Chromosome Conformation Capture Technologies (2012).

- 40 -

Popular Science Lectures • Brains@Work Academy 2009, popular science for high school students (Rotterdam) • ‘Starting a biotech company’, presentation for Netherlands Consortium for Healthy Ageing (Amersfoort, NL) • ‘Junk DNA’, seminar for Genootschap Amsterdam Other • Interviewed as opinionmaker for editorial Technology Feature in Nature: ‘Genomes in 3 dimensions’: http://www.nature.com/nature/journal/v470/n7333/full/470289a.html

Thesis advisor (promotor) of • 2011 Erik Splinter • 2013 Sjoerd Holwerda • 2013 Flore Kruiswijk (Guardavacarro group) • 2013 Laila Ritsma (van Rheenen group)

Invited speaker on meetings (2008-2014) 2014 Seminar at IGH-CNRS (Montpellier, France) 2014 CSH meeting Chromatin and Epigenetics (CSH, US) 2014 Cell symposia Transcription and Development Chicago, US) 2014 Abcam meeting Chromatin and Epigenetics (Strasbourg, France) 2014 Janelia Conference Long-range genome organization and transcription dynamics (Ashburn, US) 2013 EMBO meeting (Amsterdam, NL) 2013 Seminar at University of Manchester (UK) 2013 EMBO course on Modern Methods in Cell Biology (Heidelberg, Germany) 2013 Seminar at IMB (Mainz, Germany) 2013 Golden Helix Symposium (Amsterdam, NL) 2013 NVHG Autumn Symposium (Papendal, NL) 2013 CRG Seminar (Barcelona, Spain) 2013 French Society for Genetics (Paris, France) 2013 FEBS Congress (St Petersburg, Russia) 2013 NCMLS Technical Forum “Epigenetics and Chromatin Biology” (Nijmegen, NL) 2013 Seminar at University of Leuven (Belgium) 2012 EMBL Chromatin and Transcription (Heidelberg, Germany) 2012 CCG-UNAM Frontiers in Genomics (Mexico City, Mexico) 2012 Royal Society Discussion Meeting (London, UK) 2012 Seminar at EBI (Cambridge, UK) 2012 Seminar at Institut Pasteur (Paris, France) 2012 EMBO Conference functional genomics and systems biology (Heidelberg, Germany) 2012 Seminar at FMI (Basel, Suisse) 2011 Seminar at Max Planck (Frankfurt, Germany) 2011 WJC symposium (Copenhagen, Denmark) 2011 SKMB Gene Regulation Workshop (Lausanne, Suisse) 2011 8th European Cytogeneticists Association conference (Porto, Portugal) 2011 EMBO 2011 Nucleus meeting (Avignon, France) 2010 InteGeR meeting (Oxford, UK) 2010 Seminar at Weizmann Institute (Rehovot, Israel) 2010 Seminar at EMBL (Heidelberg, Germany) 2010 Seminar at IMPPC (Barcelona, Spain) 2010 Seminar at Center for Neurogenomics and Cognitive Research (CNCR) (Amsterdam, NL) 2010 Seminar at Netherlands Cancer Institute (Amsterdam, NL) 2010 Lausanne Genomics Days (Lausanne, Suisse) 2010 EMBO Meeting (Barcelona, Spain) 2009 Seminar at LifeTechnologies (Foster City, US) 2009 Neurogenetics seminar series, UMC (Utrecht, NL) 2009 NoE Epigenome meeting (Edinburgh, Schotland) 2009 EMBO Conference on Nuclear Structure and Dynamics (L'Isle sur la Sorgue, France) 2009 Abcam Chromatin Conference (Singapore, Singapore)

- 41 - 2009 5th course on Epigenetics in Institut Curie (Paris, France) 2009 Workshop 200 ‘Functional organization of genomes in the nucleus’(Saint-Raphael, France) 2009 BRIC (Copenhagen, Denmark) 2008 NCI Symposium on Chromosome Biology (Bethesda, US)

Grants (2008-2014) 2014 Leducq grant ‘Deciphering the Genomic Topology of Atrial Fibrillation’ € 672,000 2013 VICI Career Grant NOW Dutch Science Foundation € 1,500,000 2012 TOP grant NWO (topic: pericentromeric heterochromatin) € 755,000 2011 NanonextNL. Breast Cancer (with Philips, ErasmusMC) € 325,000 2011 EU FP7 Collaborative project on liver cancer. ‘ModHep’ € 800,000 2011 NGI PreSeed grant. 4C in DNA Diagnostics € 250,000 2011 CGC PoC grant 4C in DNA Diagnostics € 80,000 2009 KWF grant on Breast Cancer (with ErasmusMC) € 250,000 2008 ERC Starting Grant. 4C technology to study the 3D genome € 1,250,000 2008 NGI Horizon grant. Proteins folding the genome € 500,000 2008 EU International Training Network ITN ‘InteGer’ € 250,000

Patents etc. (2008-2014) • De Laat, Grosveld ‘multiplex 4C’ WO 2008/084405 • De Laat, van Min ‘4C gene sequencing’ WO 2012/005595

Previous research

In our research we are fascinated by the complex transcription regulatory landscape of our genome and we try to understand how the many regulatory DNA elements communicate with each other to ensure proper developmental gene expression. In studying this, we particularly focus on the topology of DNA, as we and others have shown that long-range gene regulation involves chromatin looping and the physical interaction of distant regulatory DNA elements. Part of our work is therefore also technology driven: we constantly aim to further improve technology for robust and quantitative detection of long-range chromatin interactions.

In our earlier work, we were the first to adapt the chromosome conformation capture (3C) technology to study DNA topology in mammals and to demonstrate that distant enhancers and promoters contact each other for gene regulation (Tolhuis, 2002; Palstra, 2003). We were also the first to demonstrate that transcription factors mediate chromatin loops (Drissen, 2004; Splinter, 2006). Further, we developed 4C technology, the first genomics technique that allows screening the genome for sites that contact a selected gene in the nucleus (Simonis, 2006).

Over the last 6 years, we have successfully applied this 4C strategy in many collaborative projects with research groups from around the world. To highlight one such exciting result: in a fruitful collaboration with the research group of Dr. Ruud Delwel based on the application of the high resolution 4C-seq technology that we developed (van de Werken et al., 2012, see below) we were able to demonstrate that a common chromosomal rearrangement in leukemia resulted in the repositioning of an enhancer away from a tumor repressor gene (which was therefore silenced) and towards a normally faraway oncogene (which is now upregulated): both events drive cancer (Gröschel, 2014). In our own work we combined 4C technology with other state-of-the-art functional genomics tools to address a number of key issues in the field. Specifically:

• we were the first to show that RNA molecules can change chromosome topology, by demonstrating that the non-coding RNA molecule Xist is necessary to fold the inactive X chromosome in female mammalian cells (Splinter et al., 2011). • we provided the first genetic evidence in mammals that an enhancer can trans-activate a gene on another chromosome (Noordermeer, 2011). • we demonstrated that cell-specific genome conformations can result in variegated gene expression (Noordermeer, 2011). • We showed that the pluripotent genome is uniquely spatially disorganized, particularly in the inactive compartment, and that key pluripotent transcription factors drive the preferential co- localization of chromosomal regions carrying high densities of their binding sites (de Wit, 2013).

- 42 -

Also, as we realized that most functionally relevant chromatin loops take place over relatively short genomic distances, between local regulatory DNA elements such as enhancers and their target promoters, we pushed the development of high resolution 4C-seq, which we believe still is the 3C strategy with the best contact resolving power (van de Werken et al., 2012). Based on the many collaborations and requests for protocols, this technology now also seems widely used in the field.

A number of years ago we realized that 3C technologies should also be very suitable to identify structural rearrangements in the genome. We explored this and modified 4C to a DNA diagnostics tool, enabling high-resolution identification of genomic rearrangements near loci of interest. This resulted a.o. in the discovery of new oncogenes in leukemia (Simonis, 2009; Homminga, 2011). Triggered by positive feedback from clinical researchers studying such rearrangements we explored the commercial potential of 4C technology as a tool to study genome integrity. We then came to a realization that a series of further changes to the protocol leads to a strategy that enables the targeted resequencing of genes. Targeted Locus Amplification (TLA), as we called the strategy, indeed enables this, with the great advantage over existing targeted sequencing approaches that it requires very little a priori knowledge of the sequence of the gene of interest. This uniquely enables TLA to robustly detect not only single nucleotide variants but also structural variants (de Vree et al., 2014). Based on the patented ideas and technologies, in 2012 de Laat (together with van Min) founded a biotech company: Cergentis (www.cergentis.com), which commercializes TLA technology and now employs several of our former lab members.

Future research

Functional genomics A major focus of the lab will continue to be the regulatory jungle of our genome, with projects aiming to understand how the many dispersed regulatory elements communicate with each other and with genes to ensure proper gene expression.

New tools for allelic multi-way DNA contact analyses. To address this issue from a new angle, we plan to move beyond the current 3C-related technologies (4C, 5C, HiC, ChIA-PET) that allow reconstructing average genomic topologies as present in the population of cells analyzed. Average structures are not very meaningful as the shape of chromatin will differ per allele, per cell, and will change over time. We therefore aim to develop strategies for the analysis of multi-way contacts at the single allele level. First proof-of-concept for an enabling 4C approach was very recently obtained, involving PacBio sequencing and allowing to score 6 or more contacts simultaneously per allele. A further variant hereof is under development, for which we will combine hybridization-based capture technologies with 3C templates to pulldown the large concatemers from selected loci, followed by PacBio sequencing. We will further optimize both strategies to address aims like - Uncovering quantitative folding properties of individual alleles in living cells - Discerning cooperative from exclusive interactions, for example between shared regulatory sequences and genes - Studying the periodicity of DNA contacts along individual chromosome fibers - Quantifying enhancer-promoter contacts in relation to transcriptional output.

We will additionally attempt to include a chromatin immunoprecipitation (ChIP) step in these strategies. The comparison between results should uncover for example which protein mediates which long- range chromatin contact.

Visualizing enhancer-promoter contacts in living cells 3C-based methods involve the crosslinking of cells and therefore give snapshots of topological features. Ideally one would like to follow long-range contacts in living cells. To accomplish this we initiated a collaboration with Kerstin Bystricky who recently published a new method for the visualization of selected genomic sites, involving the use of small (0,6-1kb) sequence platforms (anchors) for the recruitment and subsequent oligomerization of fluorescently labeled ParB proteins (Saad et al., PLoS Genetics 2014). We have invested in the creation of cell lines having a reporter gene integrated a various defined locations and are now capable of site specifically integrating its cognate strong enhancer at various distances from this gene, using the CRISPR system. We plan to combine this approach with the ANCH/ParB system, to have cell lines with a differentially labeled

- 43 - promoter and enhancer, the latter being at varying distances from the promoter. Initially we will use these cells to follow P-E interactions in real time, and relate contact frequencies measured in vivo to those measured by our 3C-based approaches. By taking into account the chromosome topological features as determined by Hi-C, we will also be able to assess the impact of topological domain organization on P-E contact frequencies. In a next series of experiments we will MS2-tag the transcripts for the simultaneous visualization of nascent transcripts, to directly follow how enhancer kissing impacts on the firing of the promoter.

Spatial Effect Variegation We recently hypothesized that cell-specific genome conformations can underlie transcriptional noise, as phenomenon we referred to as Spatial Effect Variegation (SEV) (Noordermeer et al., 2011). We will further explore this topic, using two parallel strategies. In one approach we will use generated embryonic stem cells (ESCs) carrying LacO binding platforms to accumulate a protein of interest at a defined nuclear position and ask whether gene loci elsewhere on the chromosome (in cis) or on other chromosomes (in trans) change their expression when contacting this locus. 4C profiles predict the contacting genes elsewhere in cis and in trans. Together with the group of Prof. van Oudenaarden, experts in quantitative FISH and single molecule RNA detection, we will design probes for single molecule RNA detection of these genes and investigate whether cell-specific genome conformations, and what sort of chromatin bound proteins, contribute to transcriptional noise. In the second strategy we will use the ANCH/ParB system to target distant sites on the same and on different chromosomes, and follow their contacts over cell division. This work should reveal whether there is cellular memory of spatial organization, at the level of chromosome positioning (trans contacts) and chromosome folding (cis contacts). Demonstrating the existence of spatial memory has great implications for SEV, as it would uncover a means for single cells in an otherwise identical cell population to autonomously propagate a defined expression pattern to its lineage.

Searching the genome for functionally relevant transcription factor binding sites STARR-Seq was recently developed to screen the genome for DNA segments with enhancer activity (Arnold, 2013). We are excited to modify this strategy to search for the functionally relevant binding sites (BSs) of a given transcription factor. For this, we will initially focus on the pluripotency factors Oct4, Nanog and Sox2, use available ChIP-seq knowledge on their BSs and combine STARR-Seq with SureSelect to selectively interrogate their enhancer activity. By doing this in wildtype and available conditional ko cells, we aim to determine which sites harbor enhancer activity and to what extent this depends on each of these factors. We expect this strategy to yield invaluable functional information to be superimposed on static ChIP-seq binding profiles.

NIPD for monogenetic diseases Part of our research will continue to explore the potential of our technologies for DNA diagnostic purposes. Non-invasive prenatal diagnosis (NIPD) entails the analysis of fetal DNA as present in the plasma of a pregnant mother, where it contributes up to 5% of total plasma DNA. Access mother DNA hampers robust assessment particularly of the maternal alleles transmitted to the fetus. Given the statistical challenges, NIPD currently only enables searching for the overrepresentation of entire chromosomes, i.e. scoring trisomies in the fetus. We aim to develop methodologies enabling NIPD to assess fetal carrier status also of monogenetic diseases. For this, we will use our new strategies for targeted haplotyping in mom and dad, in combination with cleverly designed SureSelect probes for the enrichment and sequencing of relevant plasma DNA sequences.

Leducq grant As part of a recently awarded collaborative Leducq project we will apply our toolbox to try to move beyond GWAS-identified associations and functionally link risk variants to target genes. We hypothesize that risk variants for atrial fibrillation (AF) affect target gene expression levels. AF is a heart disease affecting 7.5 million individuals in the US and Europe, for which medical and interventional therapies are only partially effective. We will apply our TLA-based targeted haplotyping strategy combined with allelic 4C strategies and allele-specific expression analysis to hundreds of patient-derived surgical heart biopsies. The allele-specific genomic contacts maps should identify the target genes; careful comparison of allelic expression levels will link variants to their transcriptional output. Newly identified target genes will serve as leads for targeted therapeutic interventions.

- 44 - Societal relevance and societal impact (2008-2014)

Cergentis: a biotech start-up company based on proprietary technology In our work, fundamental science driven by the further development of novel technologies has led to unanticipated breakthroughs and knowledge utilization in unrelated research fields, in this case in DNA diagnostics. It recently led to the foundation of a new bio-tech company, Cergentis, for kits and services in the DNA diagnostics. The development of the underlying proprietary technology is based on our unique technological expertise.

Background Fascinated by 3D genome organization we have modified and developed new methods to study genome topology. Specifically, we developed a technique, called 4C, that enables capturing, amplifying and sequencing of all genomic sequences spatially close to a gene (or DNA sequence) of interest. Theory dictates, and results show, that the technology primarily identifies DNA sequences neighbouring the sequence of interest on the linear chromosome template. This principle, we realized, makes 4C technology highly suitable for the targeted enrichment and complete sequencing of selected genes, i.e. for DNA diagnostics.

Now that we know the sequence of our genome and the function of many genes, DNA diagnostics is becoming increasingly more important in human health care. This is true for the detection of hereditary diseases in prenatal diagnosis, but also for the detection and treatment of ‘spontaneous’ diseases such as cancer, which arise as a consequence of DNA mutations that occur at some time during a person’s lifespan. It is increasingly recognized that success or failure (no cure, side-effects) of a given drug often depends on the exact genetic alterations carried by the individual. Personalized medicine, where the patient’s unique genetic profile dictates a personalized treatment, has therefore entered stage in the clinic and is expected to progressively replace traditional medicine (where drugs are administered without DNA analysis). Already 10% of FDA approved drugs carry a pharmacogenetic label, implying that their prescription requires prior DNA analysis of the patient, and this percentage will only increase. DNA analysis is becoming increasingly more important in medical diagnosis.

Current gene sequencing in the clinic most often entails the amplification (by PCR) and sequencing of the small parts of the gene (exons) that encode the protein sequence. Sequence alterations elsewhere in the gene, but also chromosome aberrations that introduce unknown DNA sequences into the exons, are missed by this strategy. Such currently undetectable genetic variation may account for up to 15% of all disease-causing DNA alterations, and must be identified as well. High throughput sequencing of entire genes will therefore replace current strategies in DNA diagnostics.

A few years ago most experts were predicting that whole genome sequencing would become the golden standard in DNA diagnostics. While this is still a possibility, many realize now that the targeted sequencing of only those genes relevant for treatment decisions may well be preferred over analyzing the entire genome sequence. It prevents doctors and patients from the ethical problem of how to deal with all the genetic variation found outside the genes of interest, which may have impact on other health aspects.

We anticipate that targeted gene sequencing strategies will hold the future, provided they robustly detect the entire spectrum of genetic variation in and around the genes of interest. Our modified version of 4C technology, called Targeted Locus Amplification (TLA), uniquely enables this and promises to be a very valuable and competitive tool for DNA diagnostics in the clinic.

Previous and future initiatives to realize knowledge utilization: Cergentis, a spin-off company We have demonstrated that 4C technology can be applied to detect genetic variation (Simonis et al., Nat Methods 2009; Homminga et al., Cancer Cell 2011). To secure the further development of the technique I have filed three patents on 4C technology for DNA diagnostics. With PreSeed grants from the Netherlands Genomics Initiative (NGI) and the Cancer Genomics Center (CGC), the commercial potential of our technologies was assessed and proof-of-concept for complete gene sequencing provided. Together with a business partner, Ing. M. Van Min, we attracted investors and recently founded a spin-off company: Cergentis, ‘the gene sequencing company’ (www.cergentis.com). Cergentis is based in the Utrecht area and employs 5-6 molecular biologists and bioinformaticians, including some of my most talented former students and postdocs. Cergentis pays the Hubrecht Institute to hire me as a consultant 1 day per week over a period of 2 years (contract ends Aug. 2014).

- 45 - This contract will likely be extended for one remaining year (until Aug. 2015) but now probably for only 1 day/month. In my activities for Cergentis, I guide the company’s scientific progress. Cergentis aims to have a major impact on the quality of DNA Diagnostics.

- 46 -

Jeroen den Hertog

Key publications

Choorapoikayil S, Kers R, Herbomel P, Kissa K and den Hertog J (2014) Pivotal role of Pten in the balance between proliferation and differentiation of hematopoietic stem cells in zebrafish. Blood 123: 184-190.

Bonetti M, Paardekooper Overman J, Tessadori F, Noël E, Bakkers J and den Hertog J (2014) Noonan and LEOPARD syndrome Shp2 variants induce heart displacement defects in zebrafish. Development 141: 1961-1970.

Paardekooper Overman J, Yi JS, Bonetti M, Soulsby M, Preisinger C, Stokes MP, Hui L, Silva JC, Overvoorde J, Giansanti P, Heck AJR, Kontaridis MI, den Hertog J and Bennett AM (2014) PZR coordinates Noonan and LEOPARD syndrome signaling in zebrafish and mice. Mol Cell Biol 34: 2874–2889.

Vacaru AM, den Hertog J. (2010) Serine dephosphorylation of RPTP{alpha} in mitosis induced Src binding and activation. Mol Cell Biol. 30, 2850-2861.

Faucherre, A., Taylor, G.S., Overvoorde, J., Dixon, J.E. and den Hertog, J. (2008) Zebrafish pten genes have overlapping and non-redundant functions in tumorigenesis and embryonic development. Oncogene 27, 1079–1086.

- 47 - Prof. Dr. Jeroen den Hertog Protein-tyrosine phosphatases in development

Group members Postdocs: Eriko Avsar Graduate students: Miriam Stumpf, Alexander J. Hale Technicians: John Overvoorde, Jelmer Hoeksma

Curriculum vitae group leader

Name: Jeroen den Hertog Date of birth: 20 September 1965 Nationality: Dutch

Education/positions 1983 - 1988 MSc, Utrecht University, Chemistry 1988 - 1992 PhD graduate student. Thesis: “Phosphotyrosine and neuronal differentiation. Of kinases and phosphatases”. Supervisors: Prof. Dr. S.W. de Laat and Dr. W. Kruijer, Utrecht University 1992 - 1994 Postdoc at the Salk Institute, La Jolla, CA in the lab of Tony Hunter 1994 - 1997 Project leader, Hubrecht Laboratory 1997 - Group leader, Hubrecht Laboratory/Institute 2008 - Deputy director research, Hubrecht Institute 2008 - Professor of Molecular Developmental Zoology, Leiden University

Memberships 1991 - present Nederlandse Vereniging voor Biochemie en Moleculaire Biologie 2003 - present American Society for Biochemistry and Molecular Biology 2014 - present Founding member of the Zebrafish Disease Models Society

Awards 1992 Fellowship, Dutch Cancer Society “Role of Protein-Tyrosine Phosphatases in carcinogenesis; regulation of enzymatic activity”

Other activities 2002, 2003, 2008 Co-organizer of the Dutch zebrafish meetings at the Hubrecht Institute 2014 Co-organizer of the meeting “Zebrafish: from fundamental research to industry”, Leiden, the Netherlands 2007 Co-organizer of the Europhosphatases meeting in Aveiro, Portugal 2007 Co-organizer of the 5th European Zebrafish Genetics and Development Meeting in Amsterdam 2009 Organizer of the Europhosphatases meeting in Egmond aan Zee, the Netherlands 2011 Co-organizer of the Europhosphatases meeting in Baden near Vienna, Austria

Thesis advisor for • 1997 Andre van Puijenbroek • 1997 Wouter van Inzen • 1999 Arjan Buist • 2000 Christophe Blanchetot • 2001 Astrid van der Sar • 2002 Robert Bink • 2006 Arnoud Groen • 2006 Chris Jopling • 2007 Fiona Rodriguez • 2008 Simone Lemeer • 2010 Andrei Vacaru • 2011 Mark J.L. van Eekelen

- 48 - • 2012 Vincent J. Runtuwene • 2014 Monica Bonetti • 2014 Jeroen Paardekooper Overman

Invited speaker on meetings (2008-2014) 2008 Edinburgh University, UK, host: Elizabeth E. Patton 2008 FASEB Meeting “Protein Phosphatases”, Snowmass, CO, USA 2009 Friedrich Miescher Institute, Basel, Switzerland; host: M. Bentires-Alj 2009 3rd Strategic conference of zebrafish investigators, Asilomar, CA, USA 2009 2nd international meeting on rare disorders of the Ras-MAPK pathway, Vienna, Austria 2009 Disease Modeling in Zebrafish workshop, Spoleto, Italy 2009 EUROTOX, Dresden, Germany 2009 FIGON Dutch Medicine Days 2009, Lunteren 2009 Banbury Center, Cold Spring Harbor, NY, USA 2010 Cancer Genomics Center annual meeting, Utrecht 2010 Disease Modeling in Zebrafish meeting, Boston, MA, USA 2010 FASEB meeting "Protein Phosphatases" Steamboat Springs, CO, USA 2010 Protein Phosphorylation, The Salk Institute, La Jolla, CA, USA 2010 ZF-CANCER meeting, San Sebastian, Spain 2011 PTPNET, Rehovot, Israel 2011 4th Strategic conference of zebrafish investigators, Asilomar, CA, USA 2011 4th Zebrafish disease models meeting, Edinburgh, UK 2011 Europhosphatases, Baden bei Wien, Austria 2012 Phosphatases in human disease, Melbourne, Australia 2012 Zebrafish 2012, 13th Australia and New Zealand workshop 2012 Fishing for answers: Zebrafish models of human development & disease, Cold Spring Harbor Asia Conference, Suzhou, China 2012 Cellular Signaling & Molecular Medicine, Cavtat-Dubrovnik, Croatia 2012 FASEB Meeting “Protein Phosphatases”, Snowmass, CO, USA 2013 5th Strategic conference of zebrafish investigators, Asilomar, CA, USA 2013 Aarhus University, Denmark; host: Claus Oxvig 2013 Europhosphatases 2013, Rehovot, Israel 2014 Yale University, host: Anton M. Bennett 2014 3rd European Zebrafish PI Meeting, Ein-Gedi, Israel 2014 7th Zebrafish disease models conference, Madison, WI, USA 2014 FASEB meeting on Protein Phosphatases, Nassau, the Bahamas

Grants (2008-2014) 2008 Cancer Genomics Center “Bioprospecting of fungal metabolites using zebrafish embryos”, € 75,000 2008 Collaborative Research Agreement with Schering-Plough "Cancer in zebrafish", € 500,000 2008 European Union, FP7, ZF-CANCER grant agreement no. 201439, € 398,000 2009 Netherlands Organization for Scientific Research (NWO). From Molecule to Organism Grant #819.02.021 "Shp2 signaling-mediated cell movement defects in Noonan and LEOPARD Syndrome", € 245,000 2010 Cancer Genomics Center “Anti-cancer activity of fungal secondary metabolites”, € 75,000 2012 ZonMw 205200001-1 “Bioprospecting for novel antibiotics”, € 678,000 2012 Yale/ Subward No. M12A11433 (NIH-grant) “Signaling by gain-of-function Shp-2 mutants in Noonan syndrome” (project leader: Anton M. Bennett), € 15,000

Patents etc. (2008-2014) • Patent application “Novel antibiotic” submitted on 30 July 2014

- 49 - Previous research

Protein phosphorylation on tyrosine residues is one of the most important cell signaling mechanisms, which is mediated by the antagonistic activities of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). Shortly after the first PTP was purified and cloned, PTPs have become the focus of my work. My lab has contributed to all aspects of PTP research, from crystal structure to in vivo function. By now, it is evident that PTPs have crucial roles in development and many PTPs are associated with human disease, including cardiovascular disease, diabetes and cancer. Work in my lab initially focused on the structure-function relationship of PTPs. In recent years, our focus shifted to understanding the role of PTPs in vertebrate development, using the zebrafish as a model.

Structure-function relationship of PTPs Following the identification of the first PTPs, we cloned and characterized several PTP family members in 1990, including the receptor-type PTP, RPTPalpha. We demonstrated that RPTPalpha has the potential to alter the differentiation fate of embryonal carcinoma cells and that RPTPalpha dephosphorylates and activates the PTK, Src. We contributed to a better understanding of mechanisms regulating PTPs, including dimerization, phosphorylation and oxidation. RPTPalpha dimerizes constitutively and subtle changes in rotational coupling within RPTPalpha dimers result in a reversible shift from the open, active conformation to the closed, inactive conformation. RPTPalpha is phosphorylated on Tyr789, near its C-terminus, which generates a binding site for the adaptor protein, GRB2. In addition, RPTPalpha is phosphorylated on Ser180 and Ser204 in the juxtamembrane domain, which regulates binding of its substrate, Src. PTPs are inactivated by direct oxidation of the catalytic site cysteine. In RPTPalpha, the catalytic cysteine of the regulatory membrane-distal PTP domain acts as a redox sensor and is oxidized, resulting in a conformational change, which suppresses catalytic activity of the membrane-proximal PTP domain of RPTPalpha.

RPTPalpha function in vivo We used zebrafish to investigate the function of RPTPalpha in vivo. Target-selected gene inactivation led to identification of zebrafish with nonsense mutations in ptpra well upstream of the catalytic site. Zebrafish embryos lacking RPTPalpha are embryonic lethal around 6 days post fertilization (dpf) and display gastrulation cell movement defects, reminiscent of the defects in knockdowns of the Src family kinases, Fyn and Yes. Src family kinases are substrates of RPTPalpha and dephosphorylation results in activation of the Src family kinases. Hence, the RPTPalpha knock-out phenotype is consistent with the developmental defects observed in the Src family kinase knockdown embryos. Moreover, embryos lacking functional RPTPalpha are rescued by expression of exogenous Src, suggesting that Src family kinases are the only relevant substrates of RPTPalpha.

PTPs in zebrafish To investigate the role of PTPs in vivo systematically, we identified the entire family of 47 classical PTPs in the zebrafish genome and established their expression patterns by in situ hybridization. We screened for PTPs that have a role in convergence and extension cell movements during gastrulation by morpholino-mediated knock down. Despite a few interesting hits, the morpholino screen was not very successful overall, which may be a reflection of the off-target effects of morpholinos, that are becoming evident now that more and more (engineered) knock-outs become available. Nevertheless, we identified two homologous non-receptor PTPs, Ptpn13 and Ptpn20 that have an essential role in convergence and extension cell movements.

Noonan and LEOPARD syndrome zebrafish Shp2 is an essential non-receptor PTP with two N-terminal SH2 domains that is associated with two related human syndromes, Noonan syndrome (NS) and LEOPARD syndrome (LS). Activating mutations in Shp2 cause NS, whereas inactivating mutations in Shp2 cause LS. Surprisingly, the clinical symptoms of NS and LS are similar, including short stature, cardiac defects and craniofacial defects. During adolescence, human LS patients develop café-au-lait spots on their skin, which is unique to LS. We developed a zebrafish model of NS and LS by expressing variants of Shp2 by microinjection at the one-cell stage. NS and LS zebrafish embryos display highly similar phenotypes that are reminiscent of human NS and LS, in that zebrafish embryos are shorter, display craniofacial defects and cardiac defects. The cardiac defects are caused by defective cilia function in Kupffer’s vesicle, resulting in impaired left-right asymmetry. Phosphoproteomic analysis of downstream signaling did not reveal great differences between the NS and LS variants, but we identified the adaptor protein Protein Zero Related (PZR) as the major hyperphosphorylated protein in both NS and

- 50 - LS, which is consistent with data from mouse models for NS and LS. In addition, we identified the PTK, Fer, as a hypophosphorylated protein in both NS and LS. Knockdowns of PZR and Fer in zebrafish both result in gastrulation cell movement defects, suggesting that both proteins contribute to the developmental defects in NS and LS embryos. How activating and inactivating mutations in Shp2 contribute to similar symptoms in human patients and to similar developmental defects in zebrafish embryos is a conundrum that remains to be elucidated.

Pten in zebrafish PTEN is a tumor suppressor gene that is frequently found to be mutated in human cancers. PTEN belongs to the PTP superfamily, yet it exhibits lipid phosphatase activity with high selectivity for the 3- position of phosphatidylinositol(3,4,5)triphosphate. The zebrafish genome encodes two pten genes, ptena and ptenb. To investigate the function of Pten in zebrafish, we isolated null mutants of both pten genes by target selected gene inactivation. Single homozygous mutants are viable and fertile and do not display developmental defects. Zebrafish retaining a single wild type allele develop hemangiosarcomas of endothelial origin. Double homozygous mutants are embryonic lethal and display a pleiotropic phenotype. Ptena-/-ptenb-/- embryos dislay hyperproliferation of endothelial cells, resulting in hyperbranching of blood vessels. Hematopoiesis is also affected in ptena-/-ptenb-/- mutants. Hematopoietic stem and progenitor cells (HSPCs) in ptena-/-ptenb-/- embryos colonize the caudal hematopoietic tissue where they hyperproliferate. These HSPCs engage in all blood cell lineages, but do not produce mature blood cells. Hence, in the absence of functional Pten, HSPCs hyperproliferate and arrest in differentiation. Interestingly, roughly half of the HSPCs die during endothelial hematopoietic transition (EHT) in ptena-/-ptenb-/- embryos. The surviving HSPCs have a distinct morphology. Close examination of EHT in wildtype embryos revealed that two distinct populations of HSPCs emerge from the dorsal aorta.

Biologically active secondary metabolites from fungi In 2007, we initiated a screen for novel bioactive secondary metabolites from fungi, using zebrafish embryogenesis as a read-out. We have screened 10,000 strains of fungi and found that more than 17% of these strains induce specific phenotypes in zebrafish embryos. In the process, we have generated a library of fungal secondary metabolites, which we have used in distinct bio-assays, including a screen for antibiotic activity against pathogenic, multi-resistant bacteria. In collaboration with the Medicinal Chemistry department of Utrecht University, we have set up a pipeline to purify the bioactive compounds and elucidate their chemical structure. Next to the identification of the hitherto unknown effect of several known mycotoxins on zebrafish embryogenesis, we have identified new bioactive compounds as well as a family of compounds that appears to affect BMP-signaling. Moreover, we have identified a novel chemical structure with antibiotic activity against methicillin- resistant Staphylococcus aureus (MRSA) and we have several other candidate antibiotics in the pipeline.

Future research

The focus of my lab is to elucidate the function of PTP-mediated signaling in development and disease. Emphasis is on a selection of biomedically relevant PTPs. We use zebrafish as an in vivo model, particularly because of its unique advantages, such as whole organism intravital imaging and (high throughput) screening of chemical compounds.

Regulation of PTPs by oxidation in vivo By now, it is well established that PTPs are regulated by oxidation. However, the in vivo relevance of oxidation-mediated inactivation of PTPs is lacking. In zebrafish embryos, significant levels of H2O2 have been detected upon tail fin amputation and during subsequent regeneration. We hypothesize that PTPs will be oxidized under these conditions and it is not unlikely that oxidation-mediated inactivation of PTPs is required for tail fin regeneration to occur. We will assess oxidation of PTPs upon amputation and during regeneration, and we will investigate the role of PTP oxidation in the regeneration process.

Noonan and LEOPARD syndrome zebrafish We will continue and extend our analysis of Shp2 variants in zebrafish. The observation that activating and inactivating variants of Shp2 induce similar symptoms in human patients and similar defects in zebrafish embryos suggests that these defects are not caused by altered Shp2 catalytic activity, but by

- 51 - differences in other traits of Shp2. We will directly address the role of catalytic activity and Shp2 conformational changes in the effect of NS and LS variants of Shp2. Whereas expression of Shp2 variants by microinjection at the one-cell stage phenocopied human Noonan and LEOPARD syndromes to some extent, aspects of the etiology of the syndromes were not recapitulated accurately in zebrafish. Using Crispr/CAS technology it is now feasible to introduce point mutations at will in the zebrafish genome and we will generate bona fide Noonan and LEOPARD mutant zebrafish. We have recently identified genetic mutants lacking functional Shp2a and Shp2b by target selected gene inactivation, which will help to analyze the genetic models of NS and LS. The focus of further analyses of genetic Shp2 mutant lines will be on heart development and hematopoiesis.

Pten in zebrafish Zebrafish embryos lacking functional Pten revealed that two populations of HSPCs emerge from the dorsal aorta. This observation warrants further investigation of the two populations of HSPCs by expression profiling and subsequent analysis of functional differences between the two populations of HSPCs. We have rescued the ptena-/-ptenb-/- phenotype by expression of Ptena or Ptenb. For these experiments, we used fusion proteins of Ptena or Ptenb, fused to mCherry. Much to our surprise, we observed highly characteristic subcellular relocalization of Pten from the cytoplasm to the nucleus, immediately prior to mitosis. Implications of subcellular relocalization of Pten will be investigated by analysis of mutants of Pten and by functional analysis in zebrafish mutants lacking functional Pten.

Secondary metabolites from fungi Next to the follow-up of hits from the zebrafish screen and the antibiotics screen, we have recently screened our library of 10,000 fungal secondary metabolites for Shp2 inhibitor activity. Initially, we assessed the effect of the secondary metabolites on PTP activity of the constitutively active Shp2 variant with an open conformation that was identified in NS, Shp2-D61G, and found 130 hits. A small fraction of these also inhibited wild type Shp2, but interestingly, the majority of hits did not significantly affect wildtype Shp2 activity and 19 hits actually enhanced wild type Shp2 activity 2-fold or more, suggesting these compounds affect the conformation of Shp2. We will identify the compounds that inhibit Shp2 and characterize the effect of these compounds using the zebrafish NS and LS models that we have generated.

Societal relevance and societal impact (2008-2014)

PTPs are important regulators of signaling and PTPs have been found to be associated with diseases, including cardiovascular disease, cancer and diabetes. A better understanding of the function of PTP- mediated signaling in vivo is relevant from a biomedical point-of-view. We have contributed to a better understanding over the years. Particularly the notion that loss of the tumor suppressor, Pten, enhances proliferation of HSPCs and arrests differentiation, which is relieved by treatment with the Phosphatidylinositol-3 Kinase inhibitor, LY294002, bolsters the role of Pten as tumor suppressor, particularly in leukemia.

The genes causing Noonan syndrome encode factors that act in the RAS-MAPK signaling pathway. In roughly 25% of NS cases, the gene that causes NS is not known. By resequencing of candidate genes, we have identified NRAS as a Noonan-associated gene. By exome sequencing of a case- parent trio and subsequent functional analyses, we identified the gene encoding Alpha-2 macroglobulin-like 1 as a gene that causes Noonan-like syndrome. In molecular diagnostics, NRAS and A2ML1 will from now on be analyzed to confirm NS or Noonan-like syndrome, which is of great benefit to patients.

Bacterial infections are a big threat to human health. Antibiotics have been developed as drugs against bacterial infections, but bacteria become resistant and superbugs have been identified that are resistant to all known antibiotics. The identification of novel antibiotics is imperative, because before long, patients with relatively simple bacterial infections will turn out to be untreatable. We initiated a search for new antibiotics by screening more than 10,000 strains of fungi for production of antibiotics by direct assessment of the effects of fungal extracts on multiresistant bacteria. We have identified a novel compound with antibiotic activity against gram-positive multiresistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). We have submitted a patent application covering this antibiotic compound as a first step towards further development of this compound into a drug. Several other compounds with antibiotic activity are currently in the pipeline.

- 52 -

Jacqueline Deschamps

Key publications

Neijts R, Simmini S, Giuliani F, van Rooijen C, Deschamps J. (2014). Region-specific regulation of posterior axial elongation during vertebrate embryogenesis. Dev. Dynamics 243: 88-98. van Rooijen C., Simmini S., Bialecka M., Neijts R., van de Ven C., Beck F., Deschamps J. (2012) Evolutionarily conserved requirement of Cdx for post-occipital tissue emergence. Development139: 2576-2583. van de Ven, C., Bialecka, M., Neijts, R., Young, T., Rowland, JE., Stringer, EJ., van Rooijen, C., Meijlink, F., Novoa, A., Freund, JN., Mallo, M., Beck, F. and Deschamps, J. (2011) Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone. Development 138: 3451-3462.

Bialecka M., Wilson V and Deschamps (2010) Cdx mutations causing posterior axial truncations do not impair the long term tissue progenitors in the embryonic posterior growth zone. Developmental Biology 347: 228-234.

Young, T., Rowland, J., van de Ven, C., Bialecka, M., Novoa, A., Carapuco, M., van Nes, J., de Graaff, W., Duluc, I., Freund, JN., Beck, F., Mallo, M. and Deschamps, J. (2009) Cdx and Hox genes differentially regulate posterior axial growth in mammalian embryos. Developmental Cell 17: 516-526.

- 53 - Dr. Jacqueline Deschamps Genetics of morphogenesis during axial elongation in the mouse embryo

Group members Postdocs: Shilu Amin (from 1/8/2014)

Graduate students: Salvatore Simmini (PhD defense planned for January 2015) Roel Neijts (PhD defense planned for spring 2016) Technicians: Carina van Rooijen, Joris Slingerland

Curriculum vitae group leader

Name: Jacqueline Deschamps Date of birth: 9 February 1951 Nationality: Belgian

Education/positions 1969-1973 MSc University of Brussels 1973-1978 PhD University of Brussels 1979-1983 Postdoc (Univ. Brussels, Dept. Molecular Biology, Rhode St Genèse, Belgium) 1983-1985 Postdoc in San Diego (Salk Institute, Lab Inder Verma)

1985 Hubrecht Institute

Memberships • EED (European Evolutionary Developmental Biology) • DSDB (Dutch society for Developmental Biology) • ISDB (International Society for Developmental Biology)

Other activities • Editorial: Editorial board member of Developmental Biology Guest editor for PloS Genetics

Main general organizer of international meeting • COST (consortium of European Cooperation in Science and Technology) on HOX and TALE transcription factors in Development and Disease (Egmond aan Zee, October 2013)

Scientific co-organizer of international meetings • COST (Marseille, Carry le Rouet, France) September 2011 • International Society of Differentiation/Netherlands Institute of Regenerative Medicine, Amsterdam November 2012

Initiator of the reactivation of the Dutch Society for Developmental Biology in 2010 • President of the DSDB (2010 – 2015) • Main organizer of a yearly DSDB meeting in 2011, 2012, 2013 and 2014

Seminar invitations 2008 Karolinska Institute, Stockholm, October 16 VU Genetics, Amsterdam, November 14 Basel university, November 19 2009 VU Genetics, Amsterdam, November 13 Museum d’Histoire Naturelle, Paris, March 27 MGG Rotterdam, March 24 2010 Louvain la Neuve, April 16 Leiden LUMC, June 1 Dundee University, June 4 Paris, Institut Pasteur, September 23 Geneva University, October 26

- 54 - Leuven University, November 16 2011 Marseille Luminy IBDML, March 11 VU Genetics Amsterdam, November 16 Strasbourg IGBMC, November 23 2012 Montreal University (IRCM), April 11 Vu Genetics Amsterdam, November 14 2013 University of Minho Portugal, January 16 Vu Genetics Amsterdam, November 13 2014 University of Québec (Centre de Recherche CHU)

Thesis advisor for • Ronald Vogels, 1992 • Jeroen Charité, 1994 • Eric van den Akker, 2001 • Tony Oosterveen, 2002 • Johan van Nes, 2006 • Teddy Young, July 2009 • Cesca van de Ven, December 2011 • Monika Bialecka, January 2012 • Salvatore Simmini, planned for January 2015)

Invited speaker on meetings (2008-2014) 2008: Meeting of the European Evolutionary Biology Society, Ghent, July 28-August 1 EU meeting Network of excellence SP6, Zeist, April 8-9 2009: Netherlands Society for Anatomy, Lunteren, January 9 EU meeting Network of excellence SP6, Lisbon, February 4 Symposium “Tissue Specification and Organogenesis” Lisbon, February 5-8 French Society for Life Sciences, Inst. J Monod, Paris, March 26 Meeting of the consortium of European Cooperation in Science and Technology (COST), Carmona (Sevilla), May 26-June 1 International symposium: Stem Cells in Development and Disease, Amsterdam October 27-29 2010: Netherlands Institute for Regenerative medicine (NIRM), Amsterdam, October 12-14 2011: Dutch Soc. for Dev. Biol. (DSDB renewed) inaugural symposium, Utrecht, January 21 EMBO symposium (Embryonic-Extraembryonic interfaces), Leuven, May 25-27 European COST meeting Marseille (Carry le Rouet), September 27-30 2012: International workshop on Stem Cells, Edinburgh, June 29 European Society for Evolutionary Biology, Lisbon, July 10-13 International Society of differentiation/NIRM, Amsterdam November 5-11 European COST meeting Madrid (El Escorial) November 30-December 1 2013: European COST meeting (as organizer), Egmond aan zee, NL, October 2-4 2014: EMBO workshop on Spinal cord Development, Barcelona (K. Storey organizer) October 1-4 (declined) EMBO workshop “upstream and downstream of HOX” Hyderabad, India, December 14-17 2015: EMBO workshop on Embryonic-Extraembryonic interfaces, Gottingen, May 2015

Grants (2008-2014) • NWO ALW Open Program (PhD student C. van de Ven), 2005-2009 € 216,129 • NWO ALW Open Program (PhD student S. Simmini), 2010-2014 € 245,013 • EU Network of excellence FP6 “Cells into Organs”, 2004-2009 € 200,000 • FES (Ministry of economic affairs) Bsik SCDD (Stem Cells in Development and Disease) 2005-2009 € 620,000 • FES, NIRM, Netherlands Institute of Regenerative Medicine, 2010-2015 € 874,000

- 55 - Previous research (2008 – 2014)

1- Genetic basis of vertebrate embryonic axial elongation Summary of our contribution to the research field (reviewed in Neijts et al. 2014): The vertebrate body axis extends sequentially from the posterior tip of the embryo, fueled by the gastrulation process at the primitive streak and its continuation within the tailbud. Anterior structures are generated early during gastrulation. Subsequently, nascent tissues emerging from the posterior growth zone continue to elongate the axis until its completion, in part from progenitors that are self-renewing bipotential stem cell-like cells generating neurectoderm and mesoderm. Our contribution to the field has been to demonstrate that transcription factors of several families including the homeodomain proteins Cdx and Hox are instrumental for tissue emergence during elongation of the trunk. They are at work in the posterior growth zone, and we showed that they do not affect the tissue progenitors themselves, but ensure an appropriate niche for these progenitors. We further demonstrated that this occurs by virtue of the fact that these transcription factors sustain the activity of major signaling pathways, Wnt and Fgf, at the site of tissue generation in the growth zone. We proposed an integrated model for modulation of axial extension in the mouse, with an initiation phase driven by the induction of Hox and Cdx genes by Wnt and Fgf in the posterior primitive streak, continued by a growth phase of high Cdx and trunk Hox expression, and high Wnt and Fgf, and followed by the decrease in growth after the trunk-tail transition. We proposed the latter decrease to be orchestrated by the Hox13 genes, antagonists of trunk Hox and Cdx genes and eventual terminators of axis elongation. Hereafter follows a more detailed account of these findings a) Role of Cdx genes in the maintenance of the niche for the axial progenitors of the trunk: (work started in 2007, and published by Bialecka et al., Dev Biol. 2010). The phenotype and gene expression features of the Cdx mutants that we generated have established that Cdx transcription factors are required for axial extension. We compared the contribution of axial progenitors from Gfp-labelled Cdx mutant and wild type embryos, upon grafting them to unlabeled wild type recipients subsequently cultured over the period during which truncation defects emerge in the mutants. We found that the mutant axial progenitors are rescued in a wild type environment. In other types of experiments we found that premature axial termination of Cdx mutant embryos can be partly corrected by exogenous Fgf signaling, revealing that Fgf acts downstream of Cdx, and confirming the non-cell autonomous action of Cdx on progenitor activity in the growth zone. These data imply that Cdx genes function to maintain a signaling-dependent niche for the posterior axial progenitors.

b) Cdx and Hox genes underlie elongation of axial tissues of the three germ layers by maintaining the canonical Wnt pathway active in the early precursors (Young et al., Dev Cell, 2009; van de Ven et al., Development, 2011; van Rooijen et al., Development, 2012). We found that the decrease in Cdx dosage in an allelic series of mouse Cdx mutants, as well as precocious expression of Hox paralogous 13 genes, lead to severe posterior vertebral defects, caudal neural tube defects and uro-rectal septum failure. These defects are also observed in Wnt3a null and hypomorph mutants. Phenotypic similarity, added to the fact that re-establishment of posterior Wnt pathway activity rescues the defects, lead us to propose that Cdx transcription factors act via maintaining Wnt signaling at the site of residence of tissue progenitors in the posterior growth zone of the embryo in an early phase of morphogenesis of the corresponding axial structures. These findings involve the Cdx genes upstream of canonical Wnt signaling in axis elongation, and highlight the need for correct temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They add new light on the etiology of the congenital syndromes Caudal Dysplasia or Caudal Regression in humans.

c) Integrated model of axial elongation and patterning (Young and Deschamps, Cur. Topics Genet and Devel., 2008, Young et al. Dev. Cell 2009; van Rooijen et al. Development, 2012). We could define the spatio-temporal realm of action of Cdx genes during emergence of the head to tail body axis: mouse embryos wherein all three Cdx genes are inactivated fail to generate any axial tissue beyond the cephalic and occipital primordia. Our data argue for a main function of Cdx in enforcing trunk emergence beyond the Cdx independent cephalo- occipital region, and for a downstream role of Fgf and Wnt signaling in this function. Cdx requirement for the post-head section of the axis is ancestral since it is found in arthropods as well.

- 56 - Thus Cdx and trunk Hox genes, upstream of their target Wnt and Fgf, are main players for building the trunk part of the embryonic body. Contribution during trunk growth of posterior clearance of retinoic acid (RA) by the enzyme Cyp26a1 was also inferred from our experiments. The Hox13 genes that antagonize trunk Hox and Cdx set the decrease in tissue production caudal to the trunk-tail transition and prepare for axial extension arrest.

d) Relationship between Cdx-dependent and T Brachyury –driven genetic programs during axial extension (work in progress, unpublished). T Brachyury (T Bra) has been shown to be required for embryonic axial growth since a long time, but its working mechanism has remained elusive. Its inactivation, similarly to that of Cdx genes, arrests axial extension at trunk levels. A striking property of trunk tissue generation is that it depends on higher and higher dosage of the required effectors. This is true for the transcription factors Cdx and T Bra, and for their common downstream signaling pathways Wnt and Fgf, all required in a dosage-dependent way to allow axial elongation to proceed to completion. It is still unclear how this progressively stronger dosage-dependence of the axial progenitors on signaling is molecularly regulated and imparted to the downstream program of the cell descendants. In addition, the question of the relationship between the seemingly parallel pathways driven by T Bra and by Cdx had not been dealt with yet. We set out to investigate whether the mutations in T Bra and in the most active of the Cdx genes, Cdx2, would add their effects on embryonic axial elongation, and to compare the changes in the transcriptional program downstream of these master regulators in T Bra/Cdx2 double mutant embryos and in T Bra and Cdx2 single mutants. This work is in progress. It involves a collaboration with L. Kester and A. van Oudenaarden for transcriptional analysis of early embryonic tissues via RNA-Seq experiments.

2- Role of Cdx genes in the maintenance of adult endodermal stem cells (intestinal stem cells) Summary of our contribution to the research field: While Cdx genes are instrumental in driving posterior tissue elongation in the three germ layers in a non-cell autonomous way, these genes do play a cell-autonomous role in gut endoderm morphogenesis and differentiation. a) Cdx2 determines the fate of postnatal intestinal endoderm (Stringer et al., Development 2012). Knock out of Cdx2 using the intestinal stem cell-specific Lgr5 Cre deleter allele has been performed in adult mice. Cdx2-negative intestinal crypts were observed to produce subsurface cystic vesicles while untargeted crypts hypertrophied to replace the surface epithelium. The Cdx2 null cysts exhibited signs of gastric pyloric phenotype. This work, added to data in other laboratories, suggested that Cdx2 is driving the intestinal phenotype in the adult endoderm epithelium.

b) Cdx2 is required to maintain the intestinal identity of adult crypt stem cells (ms in re-revision: Simmini et al., collaboration with H Clevers and several of his lab members, and with A. van Oudenaarden and his lab member L. Kester) The endodermal lining of the adult gastro-intestinal tract harbors stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation program and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We showed that single adult Lgr5-positive stem cells, isolated from small intestine, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2 null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. The intestinal genetic program is thus critically dependent on the single transcription factor encoding gene Cdx2.

- 57 - Future research

1- Molecular and cellular mechanism of axial elongation in the mouse embryo The available data so far strengthen the notion that axial elongation in vertebrates takes place in a sequence of distinctly regulated phases. Head/occipital, trunk, and caudal tissues are generated in a succession of windows, each of which obeys its own rules. Our plans for the next years in this respect concern the elongation of trunk tissues that we have shown to depend on several transcription factors (Cdx and Hox genes and T Brachyury) and on the appropriate control of growth signaling pathways (Wnt , Fgf and RA). a) We want to investigate the hierarchy and epistaticity of the different players modulating axial elongation of the embryonic trunk to get a deeper understanding of the genetic network underlying the different phases of post-occipital axial growth. Our strategy is to interfere with the normal dynamics of expression of effector transcription factors and signaling activities at different developmental time points, using the “Tet on” system and timed gain of function of Cdx, T Bra, Fgf, Wnt and RA clearance (collaboration with Val Wilson, Edinburgh, for the use of the T Bra rtTA allele to induce the Tet-responsive gain of function transgenes), and investigate and compare the impact of the changes on tissue elongation and morphogenesis. In combination with prolonging the expression of positive trunk effectors beyond the trunk-tail transition, we will also decrease the dosage of the axial terminator Hox13 genes by using the Hoxb13 null allele.

b) We also want to better specify the mechanism of action of the transcription factors involved in the maintenance of axial progenitor activity. Cdx2 and T Bra share a downstream stimulatory activity on axial extension via upregulating Wnt and Fgf in the posterior embryonic growth zone. However, their precise molecular downstream program at the time of trunk emergence is still unclear. To begin investigating the relationship between the downstream programs of these transcription factors, RNA-Seq will be performed using posterior parts of early somite embryos single and double mutants for T Bra and Cdx2 (collaboration with A. van Oudenaarden and Lennart Kester, Hubrecht Institute). In addition, we aim at identifying the direct genomic targets of Cdx and T Bra in the Wnt and Fgf pathways, the main active effectors of the progenitor niche, and the targets of these transcription factors in the genome in general, during the embryonic trunk elongation process. Cdx2 ChIP-Seq experiments will be performed in epiblast stem cells (EpiSCs) initially, and in E8.0 embryonic tail buds eventually, since this stage corresponds to the start of the developmental window during which Cdx2 is actively maintaining the posterior growth zone to construct trunk and tail structures. We will also perform T Bra ChIP-Seq experiments using E8.0 embryonic tailbud material. Comparing the binding profiles of these two transcription factors during the same morphogenetic event in embryos should reveal crucial information as to their mode of action. The work also involves a collaboration with M. Creighton (Hubrecht Institute) for the bioinformatic analysis.

c) We also want to investigate the impact of Cdx2 ablation on the behavior/fate of progenitors in the embryonic posterior growth zone by imaging cells and their descendants upon laser induced photoconversion of a fluorescent marker in Cdx2 null and control embryos (collaboration with A K Hadjantonakis, Sloan Kettering, NY). We will also compare the behavior/fate of posterior epiblast cells of Cdx2 null and T null mutants, to pinpoint the stage (before, during or after ingression through the streak) at which mutant progenitor cells fail to contribute descendants to elongate axial tissues.

d) Molecular mechanism of the dominant antagonism of Hox13 on trunk Hox and Cdx genes eventually causing arrest of axial extension. We will take advantage of the dedicated biological materials that we generated (a Cdx2P Hoxb13-FLAG transgenic mouse line) to reveal genomic sites bound by Hoxb13 in its activity of arresting axial elongation. The outcome of this project will be particularly interesting to compare with that of project 1.b above, dealing with mapping the binding sites and identifying direct downstream targets of Cdx2 during trunk axial extension. This will enable us to test the hypothesis according to which Hox13 genes compete with trunk Hox and Cdx genes via binding common downstream targets during embryogenesis. This study should deliver answers to the long standing question of the molecular mechanism underlying the property of “posterior prevalence”, a recurrently described but still unexplained property of Hox13 genes whereby they exert a dominant negative effect on the activity of more 3’Hox genes when co-expressed with them.

- 58 -

2- Upstream of Hox and Cdx transcription: Molecular control of initial induction of Hox and Cdx genes. Our previous work indicated that the embryonic region at the posterior end of the primitive streak of gastrulating mouse embryos is primed to induce Hox genes one full day before they are actually expressed (Forlani et al., Development, 2003). We have turned to using epiblast stem cells (EpiSCs) that represent early primitive streak epiblast, to investigate the molecular underlying of this transcriptional poising state. We determined conditions for induction of early and later HoxB genes, and Cdx genes in EpiScs, and are using these conditions to determine epigenetic changes – ChIP for histone marks - and 3D interactions - 4C, chromosome conformation capture- upon Hox induction. For this work, we collaborate with W de Laat (Hubrecht Institute) for the 4C approach, and with M. Creighton (Hubrecht Institute) for the bioinformatic analysis of Histone ChIP profiles. (Along a sideline of this study of the relationship between Hox gene expression and 3D chromosome topology in ES cells, EpiSCs and expressing embryonic tissues, we have a collaborative project with D Duboule and his postdoc Leonardo Beccali with a focus on studying the HoxD regulation in the context of their earlier work).

3- Role of Cdx genes in the maintenance of adult endodermal stem cells (intestinal stem cells) We want to elucidate the direct targets of Cdx2 in their activity of maintaining the identity of the small intestinal crypt stem cells. Depending on the sensitivity and outcome of the Cdx2 ChIP-Seq about to be performed in EpiSCs and mouse embryonic tailbuds, we will attempt to perform a Cdx2 ChIP-Seq on isolated Lgr5 positive intestinal crypt stem cells (priority of this plan relatively to the other projects described above will also depend on the progress of the competition).

Societal relevance and societal impact (2008-2014)

Our research tackles fundamental questions in developmental biology of vertebrates, using the mouse as model system. Fundamental research remains an essential level of investigations that constitutes a prerequisite to the development of clinical approaches and therapies for human disease.

Our studies on the molecular genetic basis of axis elongation in the mouse, aiming at understanding the fundamental process of axial tissue growth, are relevant for increasing knowledge on human embryogenesis. Our studies of posterior axial truncations in genetic mutant mouse embryos contribute to elucidating the etiology of a subset of human congenital birth defects such as the severe “Caudal Regression”, or the less severe hypospadias. Elucidating the genetic network of signaling pathways involved in the normal morphogenetic processes in mice, the best model system for humans, should reveal which external molecular signaling events influence human embryogenesis. This knowledge should help facilitating clinical follow up of cases where embryonic development is disturbed, and possibly contribute to failure prevention at longer term. Our work on the genetic network underlying maintenance of the undifferentiated state and identity of stem cells, both in embryos and in adults, is also of medical relevance. The parameters that we succeed in defining for the particular types of stem cells that we work on in embryos and adult mice deliver information on stem cells and their niche in general, a focus of attention in present day medical biology and therapy development . On the other hand, increased knowledge on maintenance of undifferentiated stem cells should be useful to better understand ageing, the other end of the growth process of living organisms including humans.

From a wider scientific perspective, the research data obtained on the conserved processes and genetic players driving axial tissue growth in mouse embryos have implications in the field of biology of evolution of the animal body plan.

Our work also serves as source of inspiration and motivation for young scientists, from bachelor and master students to PhD students and postdocs who participate in our research efforts. In turn, some of them may take on the mission of pursuing scientific investigations, be it at the fundamental or more applied level. In general, the teaching they receive during their training serves to increase their –and thus societal- knowledge in biology.

- 59 -

- 60 -

Niels Geijsen

Key publications (2008-2014)

Chen H-H, Welling MA and Geijsen N. (2014) Dazl limits pluripotency, differentiation and apoptosis in developing primordial germ cells. Stem Cell Reports. In press. de Wit E, Bouwman BA, Zhu Y, Klous P, Splinter E, Verstegen MJ, Krijger PH, Festuccia N, Nora EP, Welling M, Heard E, Geijsen N, Poot RA, Chambers I, de Laat W. (2013) The pluripotent genome in three dimensions is shaped around pluripotency factors. Nature 501:227-231.

Buecker C, Geijsen N. (2010) Different flavors of pluripotency, molecular mechanisms, and practical implications. Cell Stem Cell. Nov 5;7(5):559-64.

Buecker C, Chen HH, Polo JM, Daheron L, Bu L, Barakat TS, Okwieka P, Porter A, Gribnau J, Hochedlinger K, Geijsen N. (2010) A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells. Cell Stem Cell. Jun 4;6(6):535-46.

Chou Y-F, Chen H-H, Eijpe M, Yabuuchi A, Chenoweth JG, Tesar P, Lu J, McKay RDG and Geijsen N. (2008) The growth factor environment defines distinct pluripotent ground states in novel blastocyst- derived stem cells. Cell 135: 449-461.

- 61 - Prof. Dr. Niels Geijsen Stem Cell Modeling of human genetic disease

Group members Postdocs: Diego D’Astolfo and Nicolas Rivron Graduate students: Maaike Welling, Manda Arbab, Chen Chen, Axel Beier, Pieterjan Diericks and Javier Frias Aldeguer Technicians Nune Schelling, Stefan van der Elst and Peng Shang

Curriculum vitae group leader

Name: Niels Geijsen Date of birth: 15 July 1971 Nationality: Dutch

Education/positions 1989-1995 MSc Utrecht University 1995-2000 PhD, Utrecht University 2000-2004 Post-doctoral fellow, Whitehead Institute for Biomedical Research, Cambridge, MA 2004-2010 Assistant professor, Massachusetts General Hospital/ Harvard Stem Cell Institute, Boston, MA 2010-present Senior group leader, Hubrecht Institute, 2010-present Professor of Regenerative medicine, Utrech University Faculty of Veterinary Medicine

Memberships International Society for Stem Cell Research (ISSCR)

Awards 2008 NWO Vidi Award

Other activities • Lecturer, PABSELA/Harvard Medical International Stem Cell Workshop, Buenos Aires, Argentina • Lecturer in an annual course on pluripotent stem cells, University of Coimbra, Portugal • Founder and organizer, California Institute for Regenerative Medicine (CIRM)/HSCI Lead Faculty network (2006-2010) • Nordrhein Westfalen Stem Cell Network Faculty Review Committee (2013)

Thesis advisor for • Maaike Welling • Manda Arbab • Pieterjan Diericks • Axel Beier • Javier Aldeguer

Invited speaker on meetings (2008-2014) Invited speaker for international conferences and seminar series, including UC Berkeley; University of Edinburgh; University of Cambridge, Cold Spring Harbor Labs; UCLA; University of Pennsylvania; University of Connecticut; Erasmus MC; Serano Symposia International Foundation; American Society of Andrology, European society for the Study of Human Reproduction (one in 2013, 3x in 2014)

- 62 - Grants (2008-2014) • National Institutes of Health $ 1,062,450 2007-2012 RO1 (+ $ 737,907 indirect) • NWO ‘VIDI’ Award € 600,000 2008-2013 • Latran Foundation € 80,000 2010-2011 • FP7- Innovations in Medicine Initiative (IMI) € 250,000 2011-2016 • Noaber Foundation € 250,000 2011-2015 • Nierstichting € 100,000 2013-2015 • Netherlands Institute of Regenerative Medicine € 600,000 2011-2016 – Fonds Economische Structuurversterking • FP7 – IMI, EUAIMS € 250,000 2012-2016 • Marie Curie ITN – “Growsperm” € 300,000 2014-2016 • FP7 – IMI, EBiSC € 250,000 2013-2016 • Dirkzwager Assink fund € 250,000 2014-2016 • UU seed Grant € 100,000 2014-2016

Patents etc. (2008-2014) • US 13/968,852 Patent application on modulation of stem cell culture conditions to facilitate gene editing • GB1315321.8 Patent application on cell transduction technology

Previous research

Past research in the lab has focused on understanding the nature of pluripotent stem cells, the role of growth factor signaling in defining stem cell characteristics and the link between pluripotent stem cells and the germline. It integrates my expertise on signal transduction obtained during my PhD, with the stem cell methodologies I set up during my post-doctoral research, where I was the first to demonstrate that embryonic stem cells (ESC) can differentiate in vitro to generate primordial germ cells

As an independent investigator, at both MGH/Harvard Stem Cell Institute and the Hubrecht Institute, I have combined these fields and, in the past 6 years my lab has made seminal contributions to the understanding of the nature and functional properties of murine and human pluripotent stem cells (published in Cell, Nature, Cell Stem Cell and Nature Protocols).

We were the first to demonstrate that extracellular growth factors and cell signaling pathways define the epigenetic and functional properties of pluripotent stem cells, including their susceptibility to genetic manipulation (Chou et al). In particular stimulation of Wnt signaling in combination with inhibition of the MEK/ERK pathway, so-called 2i conditions results in increased robustness of the pluripotency network and in human cells makes the cell more amenable to genetic manipulation (Buecker et al a and b). More recently, we have found that one of the genes that is robustly induced by these 2i conditions is Dazl, a key regulator of germ cell development. We have explored the role and function of this gene, both in developing PGCs and in ESCs (Welling et al).

In PGCs, Dazl associates with specific mRNAs and acts as a translational inhibitor. We have demonstrated that among the targets of Dazl are important regulators of pluripotency (Sox2, Sal4 and Nanog), differentiation (Suz12) and apoptosis (Caspase 2, 7 and 9). It appears that in the developing germline, Dazl acts as a gatekeeper for pluripotency. Inhibition of important pluripotency factors prevents teratoma formation by the nascent PGCs, whereas inhibition of Suz12 assures that the PGCs do not undergo somatic differentiation. Finally, Caspase inhibition by Dazl is an elegant built-in safety switch, where failure of Dazl function leads to the expression of key caspases and triggers apoptotic cell death. In pluripotent stem cells, both in the blastocyst embryo and in cultured ESCs, Dazl has a very different role. In the developing embryo, Dazl is robustly upregulated in the last blastocyst stage, just prior to implantation (E4.5). Dazl is expressed in a small subpopulation of more naïve ESCs in serum culture conditions and is strongly upregulated in 2i culture conditions. As such, Dazl appears to mark naïve pluripotent stem cells, both in the early embryo as well as in ESCs, but Dazl is not a mere marker for naïve pluripotency, but plays an active role in the transition to this pluripotent state as well. Rather

- 63 - than acting as a translational inhibitor, our results demonstrate that in pluripotent stem cells Dazl protects mRNAs of epigenetic regulators such as Tet1 from degradation. Genetic deletion of Dazl causes failure of ESCs to induce Tet1 expression during naïve conversion. As a result, cytosine hydroxymethylation is impaired leading to a failure to induce rapid cytosine demethylation upon 2i conversion. These data provide for the first time insight in how germ cell factors regulate DNA demethylation in pluripotent stem cells and future research will be focused at exploring these regulatory mechanisms in developing germ cells as well.

In addition to exploring basic pluripotent stem cell biology, we have used pluripotent stem cells to model and investigate human genetic diseases. Using iPSCs generated from skin fibroblasts of patients with Spinal Muscular Dystrophy, we have discovered a novel pathway that we believe underlies important aspects of the disease pathology and may be involved in the motor neuron death in certain patients with ALS as well. To facilitate these studies we have also developed a novel method for the genetic manipulation and differentiation of stem- and somatic cells, based on the transduction of gene editing recombinant proteins. Given the many advantages of our novel system (viral vector free, high efficiency, precise dosage and temporal application of ectopic factors, highly improved safety, wide range of cell types and applications), it provides an ideal platform for safe gene correction in gene therapy as well as for developing better and safer stem cell reprogramming, differentiation and maturation techniques. A patent application describing this method is currently under review. Using these technologies

Future research

Our recent results have revealed a strong connection between pluripotent stem cells and the germline. Germ cells are of course the most important cells in our body, insuring species continuity. In most lower organisms, including Drosophophila, Xenopus and zebrafish, the germline is established even before fertilization, by a complex of specifying proteins and RNAs that are prelocalised in the oocyte. In contrast, in mammals germline specification is thought to occur post-implantation stages, through growth factor induction of primordial germ cells in the proximal epiblast. Conceptually, this means that while there is a continual presence of germ cells during the development of lower animals, such a continuum would not exist in mammals. Our recent data demonstrate that certain germ cell-specific genes, such as Dazl, are also expressed during preimplantation development and play an important role in establishing naïve pluripotency in the embryo. Our future work will focus on exploring whether the mammalian germline is in fact established much earlier than previously reported, and emerges from a continuing population of pluripotent cells that develop during preimplantation development. We will utilize genetic tools to trace cell lineages in the developing murine embryo, and, in collaboration with the IVF lab at UMC Utrecht and the IVF lab at the Free University of Brussels explore how pluripotency and germline specification are regulated in the human embryo.

In addition, we exploring how, in the developing murine embryo, the trophectoderm en primitive endoderm provide a pluripotent niche and directs the establishment of the early germline during peri- implantation stages. To study these interactions at the biochemical level, we are developing a new co- culture system of embryonic stem cells, trohectoderm stem cells and XEN cells.

A second research line will focus on utilizing pluripotent stem cell technologies for the study treatment of human disease. We will hereby focus on our recent identification of a new molecular pathway that appears to be misregulated in patients suffering from Spinal Muscular Atrophy (SMA). SMA is caused by a mutation or deletion of the SMN1 gene, which is embryonic lethal in all species except in humans. Humans uniquely possess a second SMN2 gene, resulting from a genomic duplication unique to our species. SMN2 can partially rescue the SMN1 gene defect, but due to a point-mutation in exon 7 of this gene, the SMN2 mRNA and protein are highly unstable and only 10% of the SMN2 mRNA results in functional and correctly folded protein. SMN is part of the mRNA splicing machinery and is found in nuclear structures called Gems. As a result of the SMN1 deletion, spinal motor neurons in patients with SMA are gradually lost after birth and 50% of the patients die before the age of 2. Current research strategies have mainly focused on enhancing or stabilizing SMN2 expression in an attempt to provide sufficient SMN activity. We have instead focused on two other, seemingly unrelated, motor neuron diseases; Spinal Muscular Atrophy with Respiratory Distress (SMARD), which displays similar early-onset motor neuron death and Amyotropic Lateral Sclerosis (ALS) in which the onset of motor neuron death typically occurs much later in life. We have found an interesting

- 64 - molecular link between SMA, SMARD and certain types of ALS and are currently preforming genetic studies demonstrating that this pathway can functionally rescue the motor-neuron death observed in these diseases. We do these both in a murine model of SMA as well as in patient-derived human pluripotent stem cells. Our future work will focus on understanding this molecular pathway and its regulation, and on the identification of potential drug-targets for the treatment of these pathologies.

An important technology that we will further develop and employ in our future studies, it the protein transduction method we have recently developed. In a manuscript, which is currently under review, we demonstrate that this technology allows highly efficient gene editing in a wide variety of somatic cells as well as pluripotent stem cells. An important strength of this methodology is the ability to introduce native proteins without the need for peptide transduction tags, that often result in mis-localization of the recombinant protein. In addition, the non-viral nature of our technology makes it an attractive tool for the clinical repair of mutations in patient (stem)cells. We will further develop this technology and establish methods for therapeutic gene editing, either through autologous stem cell transplantation, or in vivo, by local injection or whole-organ perfusion.

Societal relevance and societal impact (2008-2014)

An estimated 2-3% of births result in genetically determined abnormalities, for which there is currently no cure (source: UK Royal College of Physicians; http://www.geneticalliance.org.uk/education3.htm). Current gene therapy strategies rely on the application of viral vectors for the delivery of corrective genes. This approach bears significant risk for adverse reactions, acute immune rejection due to the high dose of injected virus and tumor formation resulting from viral integration position effects. In addition, viral gene therapies are not well suited for the introduction of large genetic elements, not much is known about potential gene silencing over time, and efficient and sustained suppression of dominant disease-causing gene is not possible.

Targeted repair of genomic mutations is the therapeutic panacea for patients suffering from genetic disease. However, existing gene therapy strategies do not repair - they merely add a functional copy of a defective gene.

Our lab has developed several technologies to facilitate stem cell gene editing. Most recently, we have discovered a method for protein-based gene editing, which resolves the issues associated with traditional gene therapy. The method allows the highly efficient delivery of gene editing proteins such as Cas/CRISPR into primary (stem) cells without the need for a viral vector. We are currently developing this technology toward a gene editing platform for the investigation or treatment of human genetic disease. We believe our technology has potential to revolutionize the treatment of genetic disease, not only to cure AAT and Hemophila B, but in application to other genetic diseases as well.

Our transduction platform has the potential to radically change research and clinical applications, as its efficiency, safety and ease of use for the introduction of biologics into primary cells, are unmatched. As is, the technology is ready for use in R&D applications, and could generate revenue in the form of licensing agreements and/or contract research. In the short term (1 year timeframe) we envision that the system can easily be commercialized in the form of siRNA transfection and gene-editing kits and as such, be distributed to the research community.

If successful, our future work will result in a proof-of-concept preclinical platform for the cure of a range of genetic diseases. Many genetic diseases have currently no cure and require life-long care and symptom relief, which is burdensome for patients, their families and their caregivers. Moreover, since the annual cost of treatment is sometimes very high, genetic diseases are an economic burden to society. Our aim is to develop a therapeutic platform that will cure or ameliorate genetic disease. It is our hope and aim that patients, their families, caregivers and society will ultimately benefit from the results of our work.

- 65 -

- 66 -

Daniele Guardavaccaro

Key publications (2008-2014)

Guardavaccaro D, Frescas D, Dorrello NV, Peschiaroli A, Multani AS, Cardozo T, Lasorella A, Iavarone A, Chang S, Hernando E, Pagano M (2008) Control of chromosome stability by the βTRCP- REST-MAD2 axis. Nature 452(7185) 365-70.

Kruiswijk F, Yuniati L, Magliozzi R, Low TW, Lim R, Bolder R, Mohammed S, Proud CG, Heck AJR, Pagano M, Guardavaccaro D. (2012) Coupled activation-degradation of eEF2K regulates protein synthesis in response to genotoxic stress. Science Signaling 5(227):ra40.

Magliozzi R, Low TW, Weijts BGMW, Cheng T, Spanjaard E, Mohammed S, van Veen A, Ovaa H, de Rooij J, Zwartkruis FJT, Bos JL, de Bruin A, Heck AJR, Guardavaccaro D. (2013) Control of Epithelial Cell Migration and Invasion by the IKKβ- and CK1α-Mediated Degradation of RAPGEF2. Dev Cell 27(5):574-85.

Magliozzi R, Kim J, Low TY, Heck AJ, Guardavaccaro D. (2014) βTrCP- and CK1-mediated degradation of Tiam1 controls the duration of mTOR-S6K signaling. J Biol Chem. In press.

Kim J, D'Annibale S, Magliozzi R, Low TY, Jansen P, Shaltiel IA, Mohammed S, Heck AJ, Medema RH and Guardavaccaro, D. (2014) USP17- and SCFβTrCP-regulated degradation of DEC1 controls the DNA damage response. Mol Cell Biol. In press.

- 67 - Dr. Daniele Guardavaccaro Ubiquitin ligases and cancer

Group members Postdocs: - Graduate students: Roberto Magliozzi, Sara D’Annibale, Jihoon Kim Technicians: -

Curriculum vitae group leader

Name: Daniele Guardavaccaro Date of birth: 5 November 1966 Nationality: Italian

Education/positions 1991 MSc, Biological Sciences (cum laude), University of Rome, Italy 1992 Military service 1998 PhD, National Research Council, Rome, Italy 1998-2004 Postdoctoral Fellow, New York University School of Medicine, New York, USA 1999-2000 Visiting scientist, Columbia University, New York, USA 2004-2008 Junior Faculty, New York University School of Medicine, New York, USA 2012 Visiting Professor, University of Modena and Reggio Emilia, Modena, Italy 2008-present Group leader, Hubrecht Institute-KNAW, Utrecht, The Netherlands

Memberships None

Awards 1993 CNR (National Research Council) 1994 Adriano Buzzati-Traverso Foundation 1995 CNR (National Research Council) 1998 IARC (International Agency for Research on Cancer) 1999 AICF (American-Italian Cancer Foundation) 2001 Susan G. Komen Breast Cancer Foundation

Other activities

Thesis advisor for 2011 Maria Augusta Sartori da Silva, Erasmus University, Rotterdam (PhD - co-supervisor) 2013 Flore Kruiswijk, CS&D Utrecht University, Utrecht the Netherlands (PhD - direct supervisor) 2014 Roberto Magliozzi, CS&D Utrecht University, Utrecht the Netherlands (PhD - direct supervisor)

Invited speaker on meetings (2008-2014) 2008 Brown University School of Medicine, Providence, RI, USA 2009 Nobel Forum, Karolinska Institutet, Stockholm, Sweden 2009 The Netherlands Cancer Institute NKI-AVL, Amsterdam, the Netherlands 2011 The 7th Salk Institute Cell Cycle Meeting, The Salk Institute, San Diego, CA, USA 2011 Gordon Research Conference, Cell Growth and Proliferation, Biddeford, ME, USA 2011 Karolinska Institutet, Department of Oncology-Pathology, Stockholm, Sweden 2013 Leiden University Medical Center (LUMC), the Netherlands 2014 University of Rome "La Sapienza" Rome, Italy 2014 University of Rome "Tor Vergata" Rome, Italy 2014 Keynote Speaker at sbv IMPROVER Jamboree, PMI and IBM, Montreux, Switzerland 2014 MD Anderson Cancer Center and Shanghai Jiao Tong University, Shanghai, China 2014 Benzon Symposium, Copenhagen, Denmark

- 68 - Grants (2008-2014) 2008 Emerald Foundation $ 150,000 2008-2013 Medical Research Council (MRC) - awarded and declined £1,677,000 (GBP) 2009-2013 Cancer Genomics Center (CGC) €1,250,000 2010-2013 Dutch Cancer Society (KWF) € 512,000 2010-2014 EU-IRG Marie Curie FP7 € 100,000

Patents etc. (2008-2014) None

Previous research

The Ubiquitin-Proteasome System (UPS) is responsible for the regulated degradation of hundreds of cellular proteins in crucial signaling and disease systems. In the last years we have been focusing on how the UPS governs cell growth and proliferation as well as cell migration and invasion. We have found that these cellular processes are controlled by an extensive crosstalk between ubiquitylation and phosphorylation. The major findings and implications of our studies are summarized below.

Regulation of cell growth and proliferation by the UPS. The kinase eEF2K controls the rate of peptide chain elongation by phosphorylating eEF2, the protein that mediates the movement of the ribosome along the mRNA by promoting translocation of the transfer RNA from the A to the P site in the ribosome. eEF2K-mediated phosphorylation of eEF2 on Thr56 decreases its affinity for the ribosome, thereby inhibiting elongation. We have recently found that in response to genotoxic stress, eEF2K is activated by AMPK-mediated phosphorylation on Ser398 (Kruiswijk et al., 2012). Activated eEF2K phosphorylated eEF2 and induced a temporary ribosomal slowdown at the stage of elongation. Subsequently, during DNA damage checkpoint silencing, a process required to allow cell cycle reentry, eEF2K is degraded by the ubiquitin-proteasome system through the SCFβTrCP ubiquitin ligase to enable rapid resumption of translation elongation. This event requires autophosphorylation of eEF2K on a canonical βTrCP-binding domain. The inability to degrade eEF2K during checkpoint silencing causes sustained phosphorylation of eEF2 on Thr56 and delays the resumption of translation elongation and mitotic entry. We have also demonstrated that mitotic entry is regulated by the phosphorylation-dependent degradation of two bHLH transcription factors, namely, TFAP4 and DEC1. While the destruction of TFAP4 is required for proper mitotic entry during normal cell cycle, DEC1 degradation is needed during recovery from the G2 DNA damage checkpoint. Indeed, failure to degrade TFAP4 leads to a slower kinetics of mitotic entry and results in a number of mitotic defects, including chromosome missegregation and multipolar spindles, which activate the DNA damage checkpoint (D'Annibale et al., 2014). Inhibiting DEC1 degradation prevents an efficient checkpoint recovery, due to a prolonged G2 phase (Kim et al., 2014). We were also able to show that DEC1 protein levels in cells are tightly controlled by a balance between ubiquitylation and deubiquitylation. During unperturbed cell cycles, DEC1 is a highly unstable protein that is targeted for proteasome-dependent degradation by SCFβTrCP in cooperation with CK1. Upon DNA damage, DEC1 is rapidly stabilized via a mechanism that requires the USP17 deubiquitylating enzyme. USP17 binds and deubiquitylates DEC1 markedly extending its half-life. Subsequently, during checkpoint recovery, DEC1 proteolysis is reestablished through βTrCP- dependent ubiquitylation. We have recently demonstrated that REST, a master transcriptional repressor that binds a 21-23 nucleotide repressor element, of which there are approximately 2000 copies in the human genome, is ubiquitylated and targeted for proteasome-dependent degradation during the G2 phase of the cell cycle to allow the activation of the mitotic spindle assembly checkpoint (Guardavaccaro et al., 2008). However, the contribution of stabilized forms of REST to cell cycle progression and cancer development has never been examined in vivo. To this aim, we have just generated knockin mice that conditionally express stabilizing mutations of endogenous REST. This animal model allows the expression of the degradation-resistant REST mutant in an inducible and tissue-specific manner. To test whether the knockin design is functional and whether recombination of the transgenic allele leads to the expression of the non-degradable REST mutant, we isolated mouse embryonic fibroblasts (MEFs) from wild type, RESTTg/+ and RESTTg/Tg embryos expressing Rosa26-CreERT2. We then assessed the amount of REST protein as well as its degradation rate before and after Cre induction with 4OH-Tamoxifen in MEFs. We have found that recombination of the REST allele by Cre activation

- 69 - after treatment with 4OH-Tamoxifen results in a dramatic stabilization of endogenous REST in RESTKi/Ki cells. We are now in the process of characterizing the phenotype of the RESTKi/Ki mice.

Regulation of cell migration and invasion by the UPS. A second research line in the group is aimed at determining the molecular mechanisms (with a special emphasis on the role of ubiquitylation and phosphorylation) controlling invasive cell migration. We have recently found that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by IKKβ and CK1α and consequently degraded by the proteasome via SCFβTrCP (Magliozzi et al., 2013). Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation- resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings have uncovered a new molecular mechanism regulating migration and invasion of epithelial cells and established a key direct link between IKKβ and cell motility controlled by Rap- integrin signaling.

Future research

Ubiquitin-mediated degradation of regulatory proteins is involved in virtually all key cellular processes. Indeed, protein degradation by the ubiquitin-proteasome system is a crucial control mechanism that ensures that molecular machines in cells are switched off or on at the right time in the specific subcellular compartment. The implication of malfunction of ubiquitin-mediated processes in human diseases and the therapeutic implications of this knowledge are also emerging. However, we are still seeing only the tip of the iceberg of the multitude of functions of the ubiquitin system in health and disease. Our knowledge of the function of E3 ubiquitin ligases, the enzymes that provide specificity to the ubiquitin-proteasome system, is still poor. Of the 650 predicted ligases, only ~20-30% have been studied at all and the number of genes that are well understood from a functional and structural perspective is much smaller. We intend to use an interdisciplinary approach including cell biological and biochemical methods as well as genetics to study the molecular mechanisms by which the ubiquitin-proteasome system governs cell growth and proliferation as well as cell migration and invasion. In particular, we will focus on how E3 ubiquitin ligases target substrates for proteasomal destruction to regulate these cellular processes. To this end, we intend to carry out: (i) A systematic genetic screen to identify E3 ubiquitin ligases implicated in the regulation of epithelial cell migration. We plan to screen an siRNA library containing siRNAs for the majority of known E3 ubiquitin ligases (HECTs and RINGs). We will screen this library employing a quantitative imaging assay of cell scattering. This assay, already used in our study of the βTrCP-mediated degradation of the Rap1 activator RAPGEF2 (Magliozzi et al., 2013), is based on the analysis of cell trajectories acquired by phase-contrast microscopy and automated cell tracking. (ii) A systematic genetic screen to identify E3 ubiquitin ligases involved in cell cycle progression and DNA damage checkpoints. We intend to screen the same E3 siRNA library and monitor cell cycle transitions by using primary cells expressing FUCCI (Fluorescent Ubiquitylation-based Cell Cycle Indicator). We will then focus on a number of E3s identified in the two screens and proceed with the characterization of their biological function. An important point would be the identification of the relevant substrate(s) of the E3 ubiquitin ligase. To this aim, in collaboration with the Netherlands Proteomics Centre, we have developed immunopurification strategies followed by mass spectrometry analysis that have been very successfully in the identification of substrates of SCF ubiquitin ligases. These strategies will be also employed to identify additional regulators such as deubiquitylating enzymes (DUBs) and protein kinases. Once the substrate of the E3 ubiquitin ligase is identified, we will perform a series of biochemical assays to characterize the molecular mechanisms by which the E3 ubiquitin ligase targets the substrate for degradation. In addition, to study the biological significance of the controlled degradation of the E3 target, we will express the mutant form that is resistant to degradation and analyze the resulting phenotype by means of appropriate cell biological assays. In specific cases, we will express the degradation-resistant mutant in the mouse and study the function of protein degradation in vivo. In an independent research line, we intend to employ an in vivo degradation assay to develop a high- throughput screen aimed at the identification of new degradation factors and pathways in the whole organism. We will use zebrafish as model organism. We will take advantage of a model GFP- substrate, i.e., ubiquitin(G76V) fused to GFP, which is efficiently ubiquitylated and degraded by the

- 70 - proteasome (no GFP signal in the wild type organism). In the presence of mutations that inhibit ubiquitin-dependent degradation, the substrate protein is stabilized resulting in the appearance of green fluorescence. We will use this system in an unbiased ENU-based forward genetic screen. Mutants will be identified by next generation sequencing and verified by morpholino and CRISPR- Cas9 technologies. Mechanistic studies will be performed as described above. Finally, in collaboration with the lab of Huib Ovaa at NKI, we will explore the possibility of modulating the activity of specific regulators of ubiquitin-dependent proteolysis as a way of developing mechanism-based therapeutics.

Societal relevance and societal impact (2008-2014)

Alterations in the ubiquitin system that occur during tumorigenesis are being uncovered, and this knowledge is starting to be exploited for both molecular diagnostics and the development of novel approaches to fight cancer. The clinical success of the proteasome inhibitor Bortezomib provided the proof-of-concept of targeting the ubiquitin-proteasome system as an anti-cancer therapeutic approach. Indeed, this drug has striking efficacy against multiple myeloma and is used for the treatment of mantle cell lymphoma. Moreover, an inhibitor of NAE-E1 (MLN4924), the enzyme needed for the activation of Cullin-RING ubiquitin ligases, has entered clinical trials for the treatment of multiple myeloma and non-Hodgkin’s lymphoma. We believe that our proposed research will increase the knowledge of the selective roles of ubiquitin ligases and their links to malignancies. At the moment, we are collaborating with the lab of Huib Ovaa to explore the possibility of modulating the activity of specific ubiquitin ligases as a way of developing mechanism-based anticancer therapeutics. We expect that inhibition of the activity of individual ubiquitin ligases will cause fewer side-effects and have a better therapeutic ratio than general inhibition of the proteasome or a whole class of ubiquitin ligases. A small molecule capable of inhibiting the ubiquitin ligase specific for a negative regulator of cell proliferation is expected to cause an increase in the cellular levels of such a negative regulator leading to a block in cellular proliferation, and consequently in cancer progression. This is especially true in those tumors that have low levels of negative regulators as a result of a "hyperactivated" ubiquitylation pathway. The examples of the Nutlins, small-molecule inhibitors that target protein-protein interaction between the tumor suppressor p53 and its ubiquitin ligase MDM2, serve as proof-of-principles of selectively targeting ubiquitin ligases. Depending on the molecular characteristics of the different tumors, one could envisage targeting specific ubiquitin ligases in different cancers as a valid approach in clinical oncology. Importantly, many SCF ubiquitin ligases promote proteasome-dependent degradation in cooperation with protein kinases for which targeted drugs have been already approved for clinical use. Elucidating the phosphorylation events and identifying the kinases involved in the degradation of a particular substrate, can lead to a rational therapeutic strategy in times relatively shorter than the ones needed to develop a novel small molecule inhibitor of an E3 ligase.

- 71 -

- 72 -

Jop Kind

Key publications (2008-2014)

Kind J, van Steensel B (2014). Stochastic genome-nuclear lamina interactions: modulating roles of Lamin A and BAF. Nucleus 5:124-30

Kind J, Pagie L, Ortabozkoyun H, Boyle S, de Vries SS, Janssen H, Amendola M, Nolen L, Bickmore WA, van Steensel B. (2013) Single-cell dynamics of genome – nuclear lamina interactions. Cell 153:178-192.

Meuleman W*, Peric-Hupkes D*, Kind J, Beaudry JB, Pagie L, Kellis M, Reinders M, Wessels L, van Steensel B. (2013) Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence. Genome Research 23:270-280.

Filion GJ*, van Bemmel JG*, Braunschweig U*, Talhout W, Kind J, Ward LD, Brugman W, de Castro IJ, Kerkhoven RM, Bussemaker HJ, van Steensel B. (2010) Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143:212-224

Kind J, van Steensel B. (2010) Genome-nuclear lamina interactions and gene regulation. Current Opinion Cell Biology 22:320-325.

- 73 - Dr. Jop Kind Spatiotemporal regulation of genomic function

Group members Postdocs: N/A Graduate students: Kim de Luca Technicians N/A

Curriculum vitae group leader

Name: Jop Kind Date of birth: 28 December 1978 Nationality: Dutch

Education/positions 1997-2002 MSc, at the University of Amsterdam 2003-2007 PhD, European Molecular Biology Laboratory, Heidelberg, Germany 2007-2008 Postdoc, European Molecular Biology Laboratory, Heidelberg, Germany 2008-2014 Postdoc, Netherlands Cancer Institute, Amsterdam, The Netherlands 2014- Hubrecht Institute

Awards • EMBO LTF • NWO Vernieuwingsimpuls (VENI)

Other activities

Invited speaker on meetings (2008-2014) 2013 4D-nucleosome workshop, Mainz, Germany 2013 MPI Dresden, Dresden, Germany 2013 EMBL Chromatin and Epigenetics meeting, Heidelberg, Germany 2014 IGBMC, Strasbourg, France

Previous research

Genome organization at the nuclear lamina Chromosomes are not just haphazard bundles of DNA randomly deposited in the 3D-space of the eukaryote nucleus. Instead, it is becoming increasingly clear that diverse nuclear processes are carefully separated in time and space. The Nuclear-Lamina (NL), a filamentous layer that lines the inner-nuclear-membrane, serves as anchor points to functionally organize the genome in three- dimensions. Those regions of the genome that associate with the NL (30-40%) are generally transcriptionally inactive; hence detailed insight in the dynamic 3D-organisation of the genome, is key in elucidating how gene-expression programs are orchestrated. Lamina-Associated-Domains (LADs) have been mapped in various species, yet, because these interaction-maps were generated in populations of cells, they provide little information on the actual dynamics and contact-frequencies of LADs in single cells. Therefore for my postdoctoral training I wished to address a simple, yet fundamental question –what are the dynamics and structural variability’s of genome-nuclear lamina (NL) interactions in single-cells? To investigate this, I conceived and developed two novel complementary single-cell techniques.

LADs are dynamic structures stochastically reshuffled after mitosis The first method involves a minimally invasive microscopy-based technique to study protein-DNA interactions in living cells. Applying this technique to study LADs in human cells, I found that LAD- dynamics are confined during interphase, yet after mitosis, the nuclear-organization is dramatically rearranged in the daughter cells. We speculate that this unanticipated plasticity in nuclear organization allows genes to provisionally “meet and greet” with the transcriptional activity in the middle of the cell and embark on transcription. This plasticity could prove pivotal for developmental genes to occasionally “monitor” the transcriptional environment e.g. during state-transitions.

- 74 -

Single-cell DamID unveils the principles of genome-nuclear lamina organization To gain insight in this remarkable diversity in chromosome architecture between individual cells, I have recently developed a second technique that faithfully captures the genome in association with the NL in single cells. Comparisons of the individual profiles reveal highly variable, yet non-random LAD- configurations between individual cells. LADs in cis, coordinately associate with the NL in a wide range of contact frequencies that are largely determined by domain-size and cooperate binding of neighboring regions. Furthermore, LAD-NL contact frequencies are inversely proportional to transcriptional output, with an increasing stochasticity in expression in LADs with intermittent genome- NL contact. Collectively, with this novel approach we gained comprehensive insight in the fundamental principles of single cell nuclear organization that will offer a framework to further studies on the effect of the probabilistic nature of genome-organization on transcriptional output.

Future research

Based on the single-cell techniques developed as a postdoctoral fellow, the main research topics in the lab will focus on elucidating the role of chromatin and nuclear architecture in gene-regulation and DNA-repair.

The role of nuclear organization in re-programming the mouse embryonic genome Cellular differentiation and re-programming are essential processes during embryonic development. Cell-fate transitions involve major changes in gene-expression programs of hundreds to thousand of genes accompanied by dramatic rearrangements of the spatial architecture of the interphase genome. In particular, hundreds of genes relocate between the nuclear lamina (a generally repressive environment) and the nuclear interior (permissive to transcription) when embryonic stem cells differentiate. Interestingly, pluripotency genes are among the genes that gain association with the nuclear lamina upon differentiation whereas genes required for more committed cellular identities re- locate towards the interior of the nucleus. Hence, detailed insight in the dynamic nuclear organization of interphase chromosomes is key in elucidating how gene-expression programs are orchestrated during cell-fate transitions. Embryonic stem (ES) cells are derived from the inner-cell-mass of the blastocyst and can be stably maintained and differentiated in large quantities. Therefore, much of our current knowledge about the involvement of chromatin and nuclear organization in maintaining pluripotency and directing differentiation is based on ES cells. In contrast, detailed information on the massive rearrangements in chromatin and nuclear organization that occur during reprogramming of the parental genomes during pre-implantation development is still lacking. Elucidating the molecular mechanism that regulates nuclear reprogramming could prove very insightful for other very related processes such as the generation of induced pluripotent cells and cancer development. Most of what we know today about the dynamic organization of pre-implantation development is derived from visual observation of fluorescent microscopy images, however we currently lack the tools to study the spatiotemporal organization and dynamics of the genome at molecular-resolution in single-cells. Lamin B1 is ubiquitously expressed and localized specifically to the nuclear lamina at all stages of pre- implantation development, which makes it an ideal marker to study nuclear organization across cell- types. By employing the m6ATracer and DamID, I plan to map and trace LAD organization at different stages of pre-implantation development in single-cells. Additionally, we wish to complement this data with single-cell interaction maps of other key chromatin types (like HP1 and Polycomb). Collectively, we strive to obtain comprehensive insight in the changing nuclear landscape, to identify key factors and decision points during the reprogramming process of early mouse development.

Profiling DNA double-stranded breaks in single cells Our genome is continuously exposed to various hazardous exogenous and endogenous DNA damaging agents. Failure to resolve DNA damage can result in the transmission of potentially harmful genomic rearrangements to future generations, which threaten cellular homeostasis and ultimately can lead to the onset of carcinogenesis. DNA double-stranded breaks (DSB) are considered the most harmful types of damage because they potentate the formation of the most comprehensive genomic rearrangements including deletions, insertions, amplifications and translocations. Such genomic alterations in turn can result in the activation of proto-oncogenes and/or loss of tumor-suppressor genes. Key in understanding the mechanism that leads to the emergence of such harmful cellular transformations, is to identify the sites in the genome that are most vulnerable to damage. Yet, upon a DNA damaging insult, every cell acquires a unique damage-profile and thus in order to map genomic

- 75 - damage-sites, cells needs to be profiled individually. Current “genomic mapping” techniques require vast numbers of cells and as such are insufficient to detect damage instances in single cells. For this project we plan to employ single-cell DamID, and adapt it to profile genome-wide the DNA-damage sites of individual cells. We will thereby distinguish between the two major DSB-repair pathways, and importantly relate the damage signatures to different cellular outcomes. Complementary to this method, we will apply the m6ATracer tool to live track the spatiotemporal behavior and repair-kinetics of individual DSB over extended time-periods. Collectively, this project proposal represents the first example of an unbiased systematic approach to uncover the nature and cellular consequences of a DNA damage insult in single-cells. This will significantly contribute to our understanding of the mechanism and fundamental principles of DNA DSB-repair.

Technique development Finally, we strive to further develop the existing toolbox and design new techniques to solve research questions that thus far could not be addressed. Recently, together with the lab of Michiel Vermeulen, we developed a quantitative proteomics technique based on pull-downs of the m6ATracer (eGFP) stably bound to different chromatin types (depending on the POI fused to Dam). The great advantage of the system is that it makes use of the strength of the existing nano-Trap (eGFP) technique, and builds on the advantages of DamID. Because the DamID m6A-DNA mark is foreign to the cell and hence is stable in time, this system makes it possible to quantitatively (SILAC-based) characterize the changing chromatin landscape over extended periods of time (up to one generation). Pilot experiments, set-up to quantify the LAD-proteome look very promising, but also other applications, such as to characterize the changing chromatin landscape after a DNA-damaging insult could also prove very powerful. These experiments combined with super-resolution-imaging of the m6ATracer (in collaboration with Kees Jalink) and DamID will provide detailed insight in the changing structure and composition of the chromatin landscape of various nuclear processes.

Societal relevance and societal impact (2008-2014)

A fundamental question in biology is how different regulatory proteins or protein-complexes are targeted to the right place at the right time. A decade of chromatin research has provided valuable information on the occupancies and targeting specificities of hundreds of components to chromatin. This has greatly advanced our knowledge of fundamental chromatin related processes in diverse cellular processes and disease, yet, this compendium of data has also shown that many proteins with diverse functions appear to bind to the same targets even in assumed homogenous cell- populations. An example of such “chromatin-crowding” is the prominent “red- chromatin” which represents an active chromatin-type in flies. A likely explanation for the apparent high occupancy of certain chromatin-types is that most proteins do not bind simultaneously at the same location in an individual cell. Therefore to gain detailed mechanistic understanding of nuclear processes, it is essential to develop novel techniques to allow the study of such processes in single cells with high temporal and molecular resolution. We have developed two complementary novel techniques to accurately map and trace protein-DNA interactions in single cells. These techniques were originally designed to study genome- NL interactions, but are readily applicable to study a multitude of genome-related processes in such diverse fields as: transcriptional-regulation, DNA-repair, stem-cell research and cancer biology. Hence, these tools will prove to be an invaluable asset to the community. Moreover, recently with a small modification in the single-cell protocol, we have accomplished to map DNA-copy-number variations in single cells. These data have a very high information-content at a modest sequencing depth. This makes it a very cost effective method to map hundreds of individual cells simultaneously we could prove a particularly valuable method to acquire insight in the onset and progression of complex and heterogeneous tumor materials and consequently will also facilitate the design of more effective therapies.

- 76 -

Puck Knipscheer

Key publications (2008-2014)

Knipscheer P, Flotho A, Klug H, Olsen JV, van Dijk WJ, Fish A, Johnson ES, Mann M, Sixma TK and Pichler A. (2008) Ubc9 sumoylation regulates SUMO target discrimination. Mol Cell 31:371-382.

Räschle M, Knipscheer P, Enoiu M, Angelov T, Sun J, Griffith JD, Ellenberger TE, Schärer OD and Walter JC. (2008) Mechanism of Replication-Coupled DNA Interstrand Crosslink Repair. Cell 134:969- 980.

Knipscheer P, Räschle M, Smogorzewska A, Enoiu M, Ho TV, Schärer OD, Elledge SJ and Walter JC. (2009) The Fanconi anemia pathway promotes replication-dependent DNA interstrand crosslink repair. Science 326:1698-1701.

Klein Douwel D, Boonen RACM, Long DT, Szypowska AA, Räschle M, Walter JC and Knipscheer P. (2014). XPF-ERCC1 acts in unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4. Mol Cell 54:460-471.

Castillo Bosch P, Segura-Bayona S, Koole W, van Heteren J, Dewar J, Tijsterman M and Knipscheer P. (2014). FANCJ promotes DNA synthesis through G-quadruplex structures. EMBO J in press.

- 77 - Dr. Puck Knipscheer Molecular mechanisms and regulation of DNA repair

Group members Postdocs: Anna Szypowska, Pau Castillo Bosch Graduate students: Daisy Klein Douwel, Wouter Hoogenboom Technicians: Merlijn Witte

Curriculum vitae group leader

Name: Puck Knipscheer Date of birth: 4 December 1974 Nationality: Dutch

Education/positions 1993 - 1999 MSc, Wageningen University 2000 - 2007 PhD (cum laude), Netherlands Cancer Institute/Erasmus University Rotterdam (Title: ‘regulation of SUMO modification’, supervisor: Prof. T.K. Sixma) 2007 - 2010 Postdoc, Harvard Medical School, Boston, USA (laboratory of Prof. J.C. Walter) 2011 - present Junior group leader Hubrecht Institute

Awards 2008 Fellowship for basic and (pre)clinical cancer research, Dutch Cancer Society (KWF) 2010 Heineken Young Scientist Award for Biochemistry and Biophysics, Royal Netherlands Academy of Arts and Sciences (KNAW) 2011 VIDI grant, Netherlands Organization for Scientific Research (NWO)

Other activities 2011 Participated in documentary on the position of woman in Science (LNVH) 2013 Jury onderwijsprijs Natuur en Techniek (KNAW) 2013 Contributed to the development of teaching material for high school students (developer: ‘De Praktijk’, Amsterdam)

Invited speaker on meetings (2008-2014) 2008 Annual Fanconi Anemia Research Fund Scientific Symposium. Eugene, USA 2009 ASM Conference on DNA Repair and Mutagenesis. Whistler, Canada 2009 Annual Fanconi Anemia Research Fund Scientific Symposium. Baltimore, USA 2010 EU-US Workshop on Dynamics of DNA repair. Smolenice, Slovakia 2011 Official opening of the International Year of Chemistry 2011. Den Haag 2011 Conference on mechanisms of aging and genome maintenance. Alaska, USA 2013 Invited seminar: Max Planck Institute of Molecular Physiology, Dortmund 2013 FEBS conference, Slovakia 2014 Invited seminar: London Research Institute, Clare Hall Laboratories

Grants (2008-2014) 2011 VIDI grant, Netherlands Organization for Scientific Research (NWO), € 704,000 2013 CancerGenomiCs.nl, Netherlands Organization for Scientific Research (NWO), € 450,000

- 78 - Previous research

My interest in understanding the molecular details of complex biological processes started during my PhD at the Netherlands Cancer Institute (in the laboratory Titia Sixma) where I investigated posttranslational modification by the ubiquitin-like modifier SUMO. Using structural, biochemical and biophysical approaches I studied the consequences of SUMO modification of two E2 enzymes and the mechanism of SUMO chain formation. During my postdoc in the laboratory of Johannes Walter at Harvard Medical School I took a different approach to study the consequences of posttranslational modifications. I contributed to the development of a unique Xenopus egg extract based system that allows the repair of DNA interstrand crosslinks (ICLs) under physiological conditions in vitro. Subsequently, I used this system to elucidate the role of the Fanconi anemia pathway, and its ubiquitylation, in the repair of this highly toxic type of DNA damage. In my laboratory at the Hubrecht Institute we continue to investigate the molecular mechanism of ICL repair. In addition, we have started to investigate the mechanism by which stable secondary DNA structures are resolved during DNA replication. Our overall aim is to make important contributions in deciphering how cells maintain genome integrity.

DNA interstrand crosslink repair ICLs covalently link the two strands of the DNA double helix together and are extremely cytotoxic, especially for rapidly dividing cells. This makes agents that induce ICLs powerful tools in cancer therapy. However, endogenous processes like lipid peroxidation and alcohol metabolism can also cause ICLs. Repair of this DNA lesion is complex, requires DNA replication and the collaboration of several DNA pathways such as nucleotide excision repair (NER), homologous recombination (HR), translesion synthesis (TLS), and the Fanconi anemia (FA) pathway. As a consequence, ICL repair is currently one of the most poorly understood DNA repair pathways. To study the molecular details of this repair pathway we use a unique method that I co-developed during my postdoc and combines Xenopus egg extracts with plasmid templates containing site-specific ICLs (Räschle, Knipscheer et al. 2008). In comparison with most cell-based assays used to study ICL repair, this system has a number of unique properties: 1) it involves only interstrand crosslinks while ICL inducing agents used in cells induce up to 95 % other types of DNA damage, 2) it allows a direct ICL repair readout, and 3) the repair reaction is synchronous which enables the analysis of consecutive steps in the process.

In this system, ICL repair occurs through a series of defined steps. First, replication forks from both sides converge 20 to 40 nucleotides before the crosslink. After a short pause, one of the forks resumes synthesis and halts again 1 nucleotide before the ICL. Endonucleolytic incisions on either side of the crosslink ‘unhook’ the ICL and lesion bypass synthesis takes place in two steps, insertion and extension. Lastly, the double stranded breaks generated during ‘unhooking’ are repaired by homologous recombination and the unhooked adduct is likely removed by excision repair. In the past years we have shown that ICL repair in this system is fully dependent on DNA replication and requires the action of the TLS polymerase Polζ and the FA proteins FANCD2, FANCP/SLX4 and FANCQ/XPF, indicating that repair follows a physiological mechanism.

Until recently it was unclear whether the FA pathway is directly involved in ICL repair. During my postdoctoral training I have used this Xenopus egg extract-based ICL repair assay to show that the FANCI-FANCD2 complex is not only strictly required for ICL repair but also plays a direct role in this process by facilitating the nucleolytic incisions that unhook the ICL (Knipscheer et al. 2009). Although this showed for the first time that the activation of the FA pathway by ubiquitylation of FANCD2 is important for a specific step in ICL repair we still did not understand how the FANCI-FANCD2 complex promoted unhooking. This was the first question I set out to answer in my own laboratory. Since FANCI-FANCD2 is not an endonuclease itself we first aimed to determine which endonucleases were responsible for making these unhooking incisions. Several structure-specific endonucleases had been implicated in ICL repair mainly based on the finding that they conferred resistance against ICL inducing agents. Using immunodepletions with antibodies against several different endonucleases we found that XPF-ERCC1 is of major importance for ICL repair and the unhooking incisions while MUS81-EME1 and FAN1 at most play a minor or redundant role. We also found that XPF-ERCC1 acts in close collaboration with an adapter protein SLX4/FANCP that is important for ICL localization. Chromatin immunoprecipitation (ChIP) experiments enabled us to follow the recruitment of these factors specifically to the ICL during repair. This way we showed that FANCD2 and its ubiquitylation promoted the recruitment of SLX4 and XPF-ERCC1 to ICLs and thereby facilitates the unhooking incisions (Klein Douwel et al. 2013). In addition to SLX4, XPF was also recently identified as an FA

- 79 - gene indicating that a major role of the FA pathway in ICL repair is the regulation of the unhooking incisions.

In addition to these targeted approaches to understand the role of specific factors in ICL repair we have recently started to use unbiased approaches to identify novel players in ICL repair. To this end we developed methods to isolate DNA-bound proteins during repair and analyse these by mass spectrometry. This work is in collaboration with M. Altelaar (Heck laboratory, University Utrecht).

Resolving G-quadruplex structures Our genome is not only threatened by damage but also by secondary structures in DNA. One particularly stable DNA structure is a G4 or G-quadruplex structure that forms in sequences containing 4 stretches of at least 3 guanines. Four guanines, one from each G-stretch, can form a planar structure stabilized by non-canonical Hoogsteen hydrogen bonds and monovalent cations. Our genome contains over 300,000 evolutionary conserved G4-sequences. Although G-quadruplexes have been implicated in several biological processes they could also cause problems during DNA replication. Consistent with this it was found that a recombinant polymerase is blocked at a G- quadruplex structure. In addition, it has been shown that in absence of specific helicases or in conditions of replication stress G4 sequences are preferentially mutated in vivo. In collaboration with the laboratory of Marcel Tijsterman (LUMC) we set out to investigate whether G-quadruplex structures directly hinder DNA replication and if so, what the mechanism is by which these structures are resolved.

We developed a method that is based on single stranded (ss) DNA plasmid templates and Xenopus egg extract. G-quadruplex structures are thought to form preferentially in ssDNA in vivo. During DNA replication, ssDNA is present at the lagging strand template but also on the leading strand template in cases of transient uncoupling of the DNA helicase from the polymerase. In both cases the approach of the growing 3’ end of the nascent strand to the G4 does not require unwinding of the DNA double helix. We mimicked this situation by replicating primed ssDNA templates in Xenopus egg extract. Using this method we found that G-quadruplex structures transiently block DNA replication even under non-compromised conditions. Nascent strand synthesis was blocked at defined positions one or two nucleotides from the G4 sequence. After transient stalling, G-quadruplexes were efficiently unwound and replicated. Depletion of the FANCJ helicase caused persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the previously described G4 sequence instability is caused by replication stalling at G- quadruplexes (Castillo Bosch et al. in press). In addition, this system gives us a unique opportunity to start to further understand the mechanisms by which these DNA structures are resolved to maintain genome stability.

Future research

The work in my laboratory in the near future will continue to focus on deciphering mechanisms that deal with replication blocking structures and lesions. Although the Xenopus egg extract system has made major contributions to our knowledge of ICL repair over the past years, many aspects of this process are still not understood. Moreover, we envision that many additional repair factors remain to be identified. In addition, we plan to use, and improve, our G-quadruplex replication assay to gain more insight into how these dangerous secondary structures are resolved. Specifically we plan to answer the following fundamental questions.

ICL repair: How are the unhooking incisions made and how is this regulated? Although we have found that a network of FA proteins is involved in regulating the incisions that unhook an ICL there are still many unanswered question. 1) How is SLX4 recruited to the ICL? This could be directly through the interaction of SLX4 with ubiquitylated FANCD2 but could also involve an additional protein or a specific DNA structure. 2) Does XPF-ERCC1 make one of the unhooking incisions or both? The strict structure specificity of this type of endonucleases suggests that the second incision requires a different nuclease. We will investigate two additional nucleases that have been implicated in ICL repair; SLX1 and SNM1A. However, there are also indications that XPF-

- 80 - ERCC1 could make both of the unhooking incisions. We will examine whether this requires dimerization through SLX4 or whether a single XPF-ERCC1 molecule could be responsible for this. What features determine ICL repair specificity of XPF-ERCC1? XPF-ERCC1 is involved in several DNA repair pathways and its mutation is associated with various genetic diseases. Recently, specific mutations in this nuclease complex were identified that cause Fanconi anemia. We aim to determine whether and how these mutations affect ICL repair, and to elucidate the features of XPF-ERCC1 that determine this ICL-repair specific function. Is chromatin remodeling required for ICL repair? Chromatin remodeling is important in many DNA repair pathways and could also play a role in ICL repair. Consistent with this, a recent paper has suggested FANCD2 to have histone chaperoning activity. We have recently shown that FANCD2 is not only recruited to the site of the ICL but it also covers the region around the ICL during repair. We will examine the potential function of FANCD2 and its spreading in chromatin remodeling during ICL repair. Are additional factors involved in ICL repair? In order to identify novel players in ICL repair we are developing methods to isolate proteins associated to ICL containing plasmids during repair. Important advantages of this approach compared to our previous chromatin isolation method is that these plasmids contain site-specific ICLs while the chromatin suffered 30-50% other types of damage. In addition, we have previously shown that ICL repair on these plasmids is synchronous, which enables us to identify repair-stage specific factors. For plasmid isolation we are optimizing a method that makes use of biotinylated nucleotides that are incorporated during DNA replication in extract. Novel ICL repair factors will first be validated in cell- based assays and then analyzed biochemically in our Xenopus egg extract based system to determine their biochemical function. What is the role of ubiquitin and SUMO modification in ICL repair? We are taking a similar mass spectrometry-based approach to identify ICL repair-associated ubiquitylation. For this we use 2 methods: 1) pull downs of flag-tagged recombinant ubiquitin added to extract, and 2) isolation of ubiquitylated proteins via K-ε-GG antibodies after trypsin digestion. Both methods are currently working in our laboratory and we have created the first datasets of ICL-repair specific ubiquitylated proteins. It is important to repeat these experiments to create a validated list of targets that we will use for functional studies. In the future we will take similar approaches to identify proteins modified with SUMO during repair.

In addition to the described approaches we are pursuing structural studies on XPF-ERCC1 and SLX4 in collaboration with Olando Schärer (Stony Brook University, USA) and Tom Ellenberger (Washington University, USA), and we are performing large-scale screens to identify XPF-ERCC1 inhibitors in collaboration with Lumir Krejci (Masaryk university).

G-quadruplexes How does FANCJ resolve G-quadruplexes? We aim to gain insights into how FANCJ resolves G-quadruplexes and how this is regulated. We will use ChIP to determine when FANCJ is recruited to G4-containing plasmids. To investigate how FANCJ is recruited we will identify FANCJ interacting factors during replication of G4-containing plasmids by mass spectrometry. What other factors are involved in resolving G4 structures? We have shown that a subset of G-quadruplex structures are resolved via a FANCJ-dependent mechanism, but how the other G-quadruplexes are resolved in our system is currently not clear. We will take two different approaches to identify additional players: 1) By testing factors that have been implicated in literature such as: Pif1, WRN, BLM, REV1 and RPA, and 2) by developing methods to isolate DNA-bound proteins on G-quadruplex containing plasmids during their replication followed by mass spectrometric anaysis. How are G-quadruplexes on dsDNA plasmids resolved? We are currently using G-quadruplexes situated on ssDNA plasmids as templates. Although we believe this system closely mimics the endogenous situation we will also investigate G-quadruplex resolving on dsDNA plasmids as dsDNA replication involves a much more complex mechanism. Therefore we will develop dsDNA substrates that enable the formation of G4 structures. With these substrates we will be able to address fundamental questions such as: Do G4 structures cause more problems when they are situated on the leading or the lagging strand? Does DNA replication stall on both strands? Does the replication fork (partially) disassemble upon collision with a G4 structure? These studies will be important to understand how these stable secondary structures can cause genome instability.

- 81 - Societal relevance and societal impact (2008-2014)

Enhancing our understanding of biomedically relevant biological processes such as DNA repair pathways is of great importance to the scientific community but also to create opportunities to advance therapeutic strategies. Our research has direct links to cancer development (Fanconi anemia, G- quadruplexes) and treatment (ICL repair).

Chemotherapy with compounds that induce ICLs is widely and successfully used but also has important drawbacks; it causes considerable side effects and patients are prone to develop resistance. In addition, the damage inflicted on the DNA induces mutations that often lead to secondary malignancies later in life. Extensive knowledge of the mechanisms by which this DNA damage is repaired is key to the development of new strategies targeting these drawbacks. Inhibiting ICL repair could allow the use of lower doses of chemotherapeutics, which could decrease side effects and secondary malignancies. In addition, such inhibitors could be powerful in targeted therapies as the synthetic lethal effect of targeting two DNA repair pathways at the same time has recently been shown to be extremely powerful in killing cancer cells. Finally, expression levels of several proteins involved in ICL repair (e.g. XPF, ERCC1 and the FA proteins) are correlated with the outcome of chemotherapy, therefore, identification if novel ICL repair factors could be of great help in personalized cancer therapy.

Our research has provided important new insights into the mechanism of ICL repair that may in the future be used to enhance efficacy of cancer treatment with DNA crosslinking agents. Moreover, our work has also increased the understanding in the mechanism of Fanconi anemia, a disease for which there is currently no treatment or cure, and could contribute towards future therapeutic strategies.

Furthermore, our studies on the resolving of G-quadruplex structures could help to explain why these stable secondary DNA structures are preferentially mutated in cancer and whether this contributes to cancer progression. In addition, G4 stabilizing molecules are being explored as anticancer drugs based on their ability to affect specific gene promoters and telomeres, and to induce DNA damage. To optimize these strategies it will be important to determine which G4 ligand most potently stabilizes G4 structures. Our system is highly suited to investigate such aspects.

I have presented our work to colleagues around the world at conferences and invited lectures. Moreover, I aim to communicate our work also to a broader audience at a regular basis. For example, I have spoken to a large general audience at the opening of the International Year of Chemistry in Den Haag and our research is used as an example in a new teaching module for high school students that is developed by ‘de Praktijk’.

- 82 -

Rik Korswagen

Key publications (2008-2014)

Yang PT, Lorenowicz M, Silhankova M, Coudreuse DYM, Betist MC, Korswagen HC. (2008) Wnt signaling requires retromer dependent recycling of MIG-14/Wls in Wnt producing cells. Dev Cell, 14:140-147.

Harterink M, Port F, Lorenowicz M, McGough I, Silhankova M, Betist M, van Weering J, van Heesbeen R, Middelkoop T, Basler K, Cullen P, Korswagen HC. (2011) A SNX3-dependent Retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biol, 13:914-923.

Ji N, Middelkoop TC, Mentink RA, Betist MC, Tonegawa S, Mooijman D, Korswagen HC* and van Oudenaarden A*. (2013) Feedback control of gene expression in the Caenorhabditis elegans Wnt pathway. Cell, 155: 869-880. *Joint senior/corresponding authors. de Groot RE, Ganji RS, Bernatik O, Lloyd-Lewis B, Seipel K, Sedova K, Zdrahal Z, Dhople VM, Date T, Korswagen HC* and Bryja V.* (2014) Huwe1-mediated ubiquitylation of Dvl defines a novel negative feedback loop in the Wnt signaling pathway. Science Signaling 7: ra26. *Joint senior/corresponding authors.

Mentink RA, Middelkoop TC, Rella L, Ji N, van Oudenaarden A and Korswagen HC. (2014) Cell intrinsic modulation of Wnt signaling controls neuroblast migration in C. elegans. Dev Cell, in press.

- 83 - Prof. Dr. Rik Korswagen Wnt signaling in development and disease

Group members Postdocs: Graduate students: Lorenzo Rella, Euclides Povoa Technicians: Marco Betist

Curriculum vitae group leader

Name: Hendrik C. (Rik) Korswagen Date of birth: 14 May 1967 Nationality: Netherlands

Education/positions 1992 MSc, Medical Biology, Utrecht University (cum laude). 1999 PhD, Netherlands Cancer Institute and University of Amsterdam (cum laude). Title of thesis: Genetic dissection of G protein-coupled signal transduction in C. elegans. Thesis advisor: Prof. dr. R.H.A. Plasterk 1998 Postdoctoral research in the group of Prof. dr. H.C. Clevers, Department of Immunology, University Medical Center Utrecht 2000 Tenure-track faculty position at the Hubrecht Institute 2005 - present Tenured faculty position at the Hubrecht Institute 2013 - present Professor of Molecular Developmental Genetics, Institute for Biodynamics and Biocomplexity (IBB), Utrecht University, Utrecht, The Netherlands.

Memberships 2010-2013 Founding member and treasurer of the Dutch Society for Developmental Biology.

Awards: 1997 Antoni van Leeuwenhoek Prize 1997 (Netherlands Cancer Institute) 1999 PhD degree with distinction (cum laude) 2002 Fellowship of the Center for Biomedical Genetics (CBG) 2006 ZonMW Vernieuwingsimpuls VIDI career grant

Committee memberships 2006-present Member of the examination and supervisory committee, University of Applied Sciences, Utrecht, the Netherlands. 2007-present Member of “Faculty of 1000” Developmental Molecular Mechanisms section. 2012-present Member steering committee of the EU FP7 MODBIOLIN consortium. 2013 Member NWO Enabling Technologies committee. 2013 Member committee Klumperman (investigating scientific integrity at UU)

Organization of recent international meetings 2010 Co-organizer of the “Wnt signaling in development and disease” meeting, Karolinska Institute, Stockholm, Sweden. 2012 Local organizer of the EMBO Conference Series meeting “30 Years of Wnt”, Egmond aan Zee, The Netherlands. 2013 Co-organizer of the 19th International C. elegans Meeting, Los Angeles, USA. 2015 Co-organizer of the 20th International C. elegans Meeting, Los Angeles, USA.

Site visits and selection committees 2010 National University of Ireland, Galway, Ireland (group of Dr. Uri Frank) 2012 DFG FOR1036 Wnt consortium, Heidelberg, Germany 2014 Selection committee for European Research Area (ERA) chair at CEITEC, Brno, Czech Republic

- 84 - Thesis advisor 2005 Damien Coudreuse (after a postdoc with Paul Nurse at Rockefeller University in New York, he is now an independent group leader at IGDR, Rennes, France). 2007 Gwen Soete (after a postdoc with Carl-Philipp Heisenberg at the Max Planck Institute in Dresden, she is now a secretary of the Scientific Council). 2008 Pei-Tzu Yang (currently postdoctoral fellow with Randall Moon, HHMI and Washington University, Seattle, USA). 2009 Jan-Willem van Ginkel (currently scientist at ORCA Therapeutics in Amsterdam). 2011 Martin Harterink (currently postdoctoral fellow with Casper Hoogenraad, Utrecht University, The Netherlands). 2014 Reinoud de Groot (currently postdoctoral fellow with Lucas Pelkmans, University of Zurich, Switzerland). 2014 Teije Middelkoop (will defend his thesis on 22 September 2014) 2014 Remco Mentink (will defend his thesis on 29 October 2014)

Invited speaker on meetings (2008-2014) 1. Cincinnati Children’s Hospital, USA, March 2008; host: Xinhua Lin. 2. Keynote at Endo-Exo meeting, French Society for Cell Biology, Batz-sur-Mer, France, May 2008. 3. icDBFG course on “Developmental Biology and Functional Genomics”, Seville, Spain, June 2008. 4. University of Southern Bohemia, Budweis, Czech Republic, July 2009; host: M. Asahina. 5. Arolla Wnt meeting, Arolla, Switzerland, August 2009. 6. Karolinska Institute, Stockholm, Sweden, October 2009. 7. IGBMC, Strasbourg, France, December 2009; host: Michel Labouesse. 8. University of Oxford, UK, January 2010; host: students biochemical society. 9. Keynote speaker at Life Sciences Meeting, University of Innsbruck, Austria, September 2010 10. Stockholm Wnt meeting, Sweden, October 2010. 11. MPI-CBG Dresden, Germany, November 2010; host: Christian Eckmann. 12. University of Nice, France, December 2010; host: Pascal Therond. 13. Inaugural Dutch Society for Developmental Biology meeting, January 2011. 14. University of Prague, Czech Republic, April 2011; host: Marie Silhankova. 15. University of Bristol, UK, May 2011; host: Pete Cullen. 16. DKFZ Heidelberg, Germany, April 2012; host: Michael Boutros. 17. Gordon Research Conference “Lysosomes and endocytosis”, Proctor Academy, Andover, New Hampshire, USA; June 2012. 18. EMBO conference series “30 years of Wnt”, Egmond aan Zee, The Netherlands; June 2012. 19. University of Zurich, Zurich, Switzerland; October 2012; host: graduate students. 20. University of Rennes, Rennes, France; November 2012; host: Damien Coudreuse. 21. CECAD Graduate Symposium, Max Planck Institute, Cologne, Germany; November 2013. 22. University of Cardiff, Cardiff, UK; February 2014; host: Trevor Dale. 23. Center for Life Sciences, Beijing, China; October 2014; host: Guangshuo Ou.

Grants (2008-2014) 2008 Research grant Koningin Wilhelmina Fonds (HUBR 2008-4114) € 480,000 Research grant NWO Genomics Horizon program (935-18-012) € 100,000 2011 NWO Middelgroot (co-applicant) € 300,000 2013 Research grant NWO ALW Open Program € 240,000 2013 NWO graduate program QBIO-CLS (co-applicant with IBB groups UU) 2014 NWO Middelgroot (co-applicant) € 663,000

Grants by group members 2008 Boehringer Ingelheim Foundation PhD fellowship (Drs. M. Harterink) NWO VENI (200 kE) (Dr. M. Lorenowicz)

Patents etc. (2008-2014) None

- 85 - Previous research

Wnt proteins are members of an evolutionarily conserved family of signaling proteins with important functions in development, adult tissue homeostasis and disease. At the cellular level, Wnt proteins can trigger a wide variety of response, ranging form cell fate specification and proliferation to cell polarization and migration. We are studying the signaling mechanisms that mediate these different effects of Wnt. We have mostly concentrated on the following topics:

1. The mechanism of Wnt secretion:

Using a combination of genetic studies in C. elegans and cell biological experiments in mammalian tissue-culture cells, we have discovered that Wnt proteins require a specialized secretion pathway to be released from Wnt producing cells. A central player in this secretion pathway is the Wnt binding protein Wntless (Wls), which binds Wnt in the endoplasmic reticulum (ER) and escorts it through the Golgi network to the plasma membrane for release. We found that Wls is a limiting component in this pathway and that it needs to be recycled back to the trans-Golgi network (TGN) to take part in new rounds of Wnt secretion (Yang et al., Dev Cell 2008). This is mediated through a retrograde trafficking pathway that involves internalization of Wls from the plasma membrane and retrieval of Wls from endosomes to the TGN, a trafficking step that is mediated by the retromer complex. Loss of retromer function leads to a strong reduction in Wnt secretion and induces various Wnt signaling related defects (Coudreuse et al., Science 2006).

Further analysis of the retromer dependent endosome to TGN transport of Wls revealed that it is mediated by a novel retromer pathway (Silhankova et al., EMBO J 2010; Harterink et al., Nature Cell Biol 2011). In this pathway, the cargo-selective subunits of the retromer interact with the sorting nexin family member SNX3 and sort Wls into vesicular transport carriers that are morphologically distinct from the tubular transport carriers that are formed by the classical retromer complex. We hypothesize that this specialized retrieval pathway may enable Wnt producing cells to uncouple the transport of Wls from other retromer cargo proteins. Such uncoupling may be necessary to achieve the tight regulation of Wnt secretion that is necessary for normal development and adult tissue homeostasis.

2. Regulation of canonical Wnt/β-catenin signaling:

Canonical Wnt/β-catenin signaling controls the migration of the C. elegans QL neuroblast descendants by inducing expression of the target gene mab-5. In wild type animals, mab-5 shows very little variability in expression levels. To study how such low variability is achieved, we collaborated with the group of Alexander van Oudenaarden to combine single cell transcript counting (single molecule mRNA FISH) with mutant analysis (Ji et al., Cell 2013). Interestingly, we found that when certain Frizzled Wnt receptors are mutated, the variability in mab-5 expression is strongly increased. Further examination of various Wnt pathway mutants led to the discovery that there is extensive cross-talk within the Wnt pathway, with embedded interlocked positive and negative feedback loops that dampen the variability in target gene expression. These results highlight the influence of gene network architecture on expression variability and implicate feedback regulation as an effective mechanism to ensure developmental robustness.

In a parallel line of research, we discovered that the HECT-domain containing ubiquitin ligase Huwe1 functions as an evolutionarily conserved negative regulator of Wnt/β-catenin signaling (de Groot et al., Science Signal 2014). Huwe1 binds to and ubiquitylates the cytoplasmic Wnt pathway component Dishevelled (Dvl). Importantly, we found that instead of targeting Dvl for degradation, Huwe1 inhibits the multimerization of Dvl that is required for signaling activity.

3. Mechanism of Wnt dependent cell migration:

Wnt proteins are key regulators of cell migration and axon guidance. In C. elegans, the migration of the QR neuroblast descendants requires multiple Wnt ligands and receptors. We found that the migration of the QR descendants is divided into three sequential phases that are each mediated by a distinct Wnt signaling mechanism (Mentink et al., Dev Cell 2014). First, long-range anterior migration is mediated by parallel-acting MOM-5/Frizzled and CAM-1/Ror2 dependent non-canonical Wnt pathways. Second, once the QR descendants reach their final anterior position, migration is stopped by activation of canonical Wnt/β-catenin signaling. Finally, the short-range dorsoventral migration that

- 86 - places the QR descendants at their ultimate positions is dependent on a planar cell polarity (PCP) related Wnt pathway. Interestingly, we found that Wnt ligands do not act instructively in this process. Instead, our results show that the QR descendants switch between these signaling pathways by temporally regulating the expression of a Frizzled Wnt receptor. Cell intrinsic timing of receptor expression is also an important guidance mechanism in mammalian nervous system development, but the mechanism of this temporal regulation is still unknown. Our work provides a powerful in vivo model to study cell intrinsic timing mechanisms and Wnt pathway cross talk at single cell resolution.

Future research

Our recent work on Wnt dependent cell migration provides a solid foundation for future studies on non- canonical Wnt signaling mechanisms in cell migration, Wnt pathway cross-talk and cell intrinsic timing mechanisms. In addition, we will continue our work on Wnt secretion and Huwe1.

1. Non-canonical Wnt signaling in cell migration:

The migration of the QR descendants is mediated by two parallel acting non-canonical Wnt pathways. The first pathway involves the Frizzled Wnt receptor MOM-5 and is required for cell motility, while the second pathway, which depends on the Wnt binding receptor tyrosine kinase CAM-1/Ror2, is required for the persistent polarization of the QR descendants. Both Frizzled receptors and Ror2 are also required for mammalian cell migration, but the non-canonical Wnt signaling pathways that are triggered by these receptors are still largely unknown. More detailed understanding of these signaling pathways is of clinical importance, because upregulation of Ror2 signaling is strongly correlated with invasion and metastasis in a wide range of human cancers.

We will use the migration of the QR descendants as a powerful model system to study these signaling mechanisms at single cell resolution in vivo. In our toolkit we have methods for genetic screens, CRISPR mediated gene targeting and high resolution live cell confocal imaging of the migration process in wild type and mutant animals. In addition, we will use an in vivo protein-tagging approach to identify binding partners of CAM-1, MOM-5 and other downstream pathway components. A key aim of this project will be to translate our results to mammalian Wnt dependent cell migration and cancer metastasis. In collaboration with the group of Jacco van Rheenen at the Hubrecht Institute, we will therefore set up in vitro cell migration assays and establish mouse models for Wnt and Ror2 dependent tumor cell metastasis.

2. Cross-talk between canonical and non-canonical Wnt signaling in cell migration:

When the QR descendants reach their final position, migration is stopped by a switch to canonical Wnt/β-catenin signaling. In this project, we will study how canonical Wnt signaling terminates the MOM-5/Frizzled and CAM-1/Ror2 dependent migration of the QR descendants. Mechanistic insight into this cross-talk is interesting from a medical perspective, as there is a similar antagonistic relationship between canonical Wnt/β-catenin signaling and Ror2 dependent invasion and metastasis in cancers such as melanoma. To study the cross-talk mechanism, we will first examine the changes in gene expression that are induced by the switch to canonical Wnt/β-catenin signaling. The transcriptome of the QR descendants will be determined through a cell specific mRNA tagging and pull-down approach and will be performed in wild type as well as in gain and loss of function mutants of the canonical Wnt/β-catenin pathway. Wnt regulated genes that are required for migration termination will be examined in detail using the assays developed in project 1.

3. Cell intrinsic timing mechanisms:

The switch from non-canonical to canonical Wnt/β-catenin signaling that terminates the anterior migration of the QR descendants is mediated through a cell intrinsic timing mechanism. At a specific time point in the migration process, the QR descendants induce expression of mig-1, a Frizzled type Wnt receptor that is necessary and sufficient to stop the migration. Cell intrinsic timing of receptor expression has also been observed in mammalian axon guidance, but the mechanism of this temporal regulation is not known. One possibility is that the expression of the receptor is controlled through the time dependent decay of a repressor. To test this hypothesis, we will scan the mig-1 promoter sequence for regions that are required for transcriptional repression during the migration phase. The

- 87 - identification of such a region may in turn direct us to the transcription factor that mediates the repressive effect. As a more general approach, we will study the gene expression profile of the QR descendants at different stages in the migration process to determine whether there are other temporally regulated genes. We will isolate single QR descendants from dissociated larvae and in collaboration with the group of Alexander van Oudenaarden we will perform single cell transcriptomics. Expression profiles will be verified in vivo using single molecule mRNA FISH and genes showing interesting expression dynamics will be followed up using the assays developed in project 1.

4. Wnt secretion pathway:

We have discovered that Wls retrieval is mediated through a novel retromer pathway. This pathway sorts Wls into vesicular carriers that are morphologically distinct from the tubular carriers that are formed by the classical retromer pathway. An important mechanistic question is how these vesicles are formed, as none of the components of the Wls specific retromer complex have membrane deforming activity. In collaboration with the group of Peter Cullen in Bristol, we have found that the Wls specific retromer complex binds to a lipid flippase and that the activity of this flippase is required for Wls retrieval. In this project we will test the model that lipid translocation by the flippase provides the membrane bending activity that is necessary for vesicle formation.

5. Role of Huwe1 in colon cancer:

Most colon tumors contain mutations that activate the canonical Wnt/β-catenin pathway. We have shown that the ubiquitin ligase Huwe1 functions as a negative regulator of canonical Wnt/β-catenin signaling. Interestingly, sequencing of tumor samples (COSMIC database) has shown that Huwe1 is frequently mutated in colon cancer, indicating that it may have a tumor suppressive function in this tissue. We will test this possibility in collaboration with Owen Samson at the Beatson Institute in Glasgow.

Societal relevance and societal impact (2008-2014)

Deregulation of Wnt signaling plays a major role in cancer. Mutations that constitutively activate the Wnt/β-catenin pathway are found in the majority of colon tumors and are prevalent in a wide-range of other tumors as well. Furthermore, there is a clear link between the non-canonical Wnt signaling mechanisms that we are studying and cancer progression. Prime examples are melanoma, gastric cancer and glioma, where activation of the Ror2 signaling pathway shows a strong causal relationship with tumor cell metastasis and poor patient survival. Despite the importance of Ror2 signaling in metastasis, the mechanisms that couple Wnt signaling to directed cell migration are still largely unknown. The objective of our research is to gain a deeper understanding of canonical and non- canonical Wnt signaling mechanisms, with the ultimate aim of identifying new entry points for Wnt pathway modulation in cancer treatment. This latter aspect will be pursued in collaboration with Merck Serono in Germany.

- 88 -

Catherine Rabouille

Key publications (2008-2014)

Schotman H, Karhinen L, and Rabouille C. (2008) dGRASP mediated non-conventional integrin secretion is required for epithelial integrity. Dev. Cell 14:171-182.

Zacharogianni M, Kondylis V, Tang Y, Xanthakis D, Farhan H, Fuchs F, Boutros M and Rabouille C. (2011) ERK7 is a negative regulator of protein secretion in response to amino acid starvation by modulating Sec16 membrane association. EMBO J. 30:3684-700.

Weil TT, Parton RM, Herpers BH, Soetaert J, Veenendaal T, Xanthakis D, Dobbie I, Halstead J, Hayashi R, Rabouille C* and Davis I.* (2012) Drosophila patterning is established by differential association of mRNAs with P bodies. Nat. Cell Biol. 14:1305-13

Bellouze M, Schäfer MK, Buttigieg D, Baillat G, Rabouille C and Haase G. (2014) Golgi fragmentation in pmn mice is due to a defective ARF1/TBCE cross talk that coordinates COPI vesicle formation and tubulin polymerization. Hum. Mol. Genet. pii: ddu320.

Grieve A and Rabouille C (2014) Extracellular cleavage of E-cadherin promotes epithelial extrusion. J. Cell Science. 127:3331-46

- 89 - Dr. Catherine Rabouille Secretion regulation

Group members Postdocs: - Graduate students: Margarita Zacharogianni and Angelica Aguilera Technicians: Marinke van Oorschot

Curriculum vitae group leader

Name: Catherine Rabouille Date of birth: 22 February 1962 Nationality: French

Education/positions 1984-1985 MSc biochemistry (Paris, France) 1985-1988 PhD biochemistry (Compiegne, France) 1989-1999 Postdoc(s) (Boston, Utrecht, London, Oxford) 1999-2002 Junior group leader (Edinburgh, UK) 2002-2010 Associate professor, UMC Utrecht (NL) 2010-present Senior group leader, Hubrecht Institute

Memberships American Society for Cell Biology

Other activities • Grant committee (Wellcome Trust, CNRS/INSERM) • Meeting organization:  Organisation and chair of Minisymposium “Structural proteins of the Golgi apparatus” of the EMBO meeting. Amsterdam. NL  Organisation and chair of the minisymposium “Adhesion” at the 21th European Drosophila Research Conference, Nice, 18-21 Nov 2009.  Organisation committee of the Annaberg EMBO meeting “Protein and lipid sorting”. Austria.  Organisation of the ESF meeting on “Cell Polarity and membrane traffic”. Poland

Invited speaker on meetings (2008-2014) 2008 • Key-note speaker for the Italian Association of Cell and Developmental Biology, Sienna, Italy • FEBS Golgi meeting, Pavia, Italy, • EMBO conference series “At the joint edge of cellular microbiology & cell biology”. Villars-sur-Ollon, Switzerland, • NVvM minisymposium “Imaging cellular membrane trafficking”. Lunteren, NL 2009 • ESF-EMBO Meeting on Cell Polarity and Membrane Traffic. Sant Feliu de Guixols, Spain. • Golgi special meeting on “how proteins move through the Golgi”. Barcelona, Spain • FASEB/EMBO Summer Research Conference on "Intracellular RNA Transport and Localized Translation". Saxtons River, VT. • EMBO meeting. Amsterdam, NL • 21th European Drosophila Research Conference, Nice, France

- 90 - 2010 • Annaberg EMBO conference “Protein and lipid sorting”. Godegg, Austria. • ESF worshop “ER function”. Girona, Spain. 2011 • Key note speaker of the Swiss fly meeting, Bern, Switzerland. • International graduate school “Quantitative biology in protein transport”, Heidelberg, Germany. • EMBO meeting “mRNA localization and translational control”. Il Ciocco. Barga, Italy. • Japanese Biochemical Society meeting, Minisymposium “Conservation and differentiation of organelle structure and function”, Kyoto, Japan. • Nederlands Biophysics meeting, NVvM minisymposium “Microscopy of Cells”. Veldhoven, NL. 2012 • ESF/EMBO meeting “membrane traffic and cell polarity”, Pultusk, Poland. • Minisymposium of ASBMB, San Diego, CA. 2013 • ASBMB symposium on “The Multitasking Endoplasmic Reticulum in Health and Disease”, Airlie, Washington, • Membrane traffic retreat Pittsburgh, • EMBO RNA meeting, Niagara on the lake, Ontario, Canada. • Gordon conference “Molecular membrane biology”, Andover, NH. • Key Note lecture at the Young Investigator meeting “protein trafficking”. Bristol, UK. • Minerva meeting “Protein and mRNA localization” Rehovot, Israel. 2014 • Annaberg EMBO Workshop “Protein and lipid trafficking” Godegg, Austria. • EMBO workshop “Trafficking and signaling”, Konstanz, Germany. • ESF/EMBO meeting “membrane traffic and cell polarity”, Pultusk, Poland. • British society for Cell Biology/Developmental Biology special meeting “The dynamic Cell”. Cambridge, UK.

Grants (2008-2014) 2008 TOP subsidie ZOnMW. € 675,000. Unconventional integrin secretion: Role in epithelium integrity and tumour formation. 912080241 2009 ESF collaborative research project: € 222,000. Unconventional protein secretion (UPS). 2012 Open ALW NWO: € 248.000. 822-020-016

Patents etc. (2008-2014) -

Previous research

Morpho-functional characterization of the secretory pathway in Drosophila Since I established my group (in 1999, as an MRC fellow at The Wellcome Trust Centre for Cell Biology, Edinburgh, UK), I have been interested in understanding the molecular mechanisms behind the functional organisation of the secretory pathway in Drosophila. In the last 5 years, we have elucidated the role of a large hydrophilic protein, Sec16 in the biogenesis of the ER exit sites, the first organelle of the secretory pathway (Ivan et al, 2008). We have then performed a targeted RNAi screen to identify Sec16 receptor at the ER (Kondylis et al, 2011). The receptor has not been but it has led the foundations of the project on stress (see below).

Membrane traffic in development: A tale of proteins and mRNAs. We have also used Drosophila to investigate the role of membrane traffic (and the secretory pathway) in development by combining fly biology and genetics to (electron) microscopy. During the last 6 years, we have pursued this, but also used mammalian systems. We have first focused on understanding the relationship between the localization of key proteins in the Drosophila oocyte, such as Gurken and Bicoid, and their mRNAs. To do so at the appropriate resolution, we have developed a novel protocol to visualise both endogenous and injected RNA at the ultrastructural level in the Drosophila oocyte (ISH-IEM, Herpers et al, 2010). In collaboration with the group of Ilan Davis (Oxford, UK), we have then used this technique and shown that gurken RNA is anchored in large cytoplasmic structures called sponge bodies in a dynein

- 91 - dependent manner in the Drosophila oocyte (Delanoue et al, 2007). We were then the first to show that the sponge bodies are in fact oocyte P-bodies, and that the differential localisation of mRNA within these structures modulates their translation (Weil et al, 2012). We have also used our knowledge on Golgi biology to elucidate the long lasting question regarding the mechanism behind the Golgi fragmentation observed in ALS. In collaboration with Georg Haase (Marseilles, France), we have used an ALS-like pathology (pmn mice) and unraveled a novel cross- talk between microtubule and the COPI coat (Bellouze et al, 2014). Recently, in collaboration with Eva van Rooij (Hubrecht), we investigate how antimRs enter the heart cells (in culture) and are stored intracellularly to inhibit microRNAs. This is done in basal and stressful conditions.

Secretion adhesion molecules. During the course of our experiments in Drosophila and mammalian cells, we have investigated the role of gap junction proteins Innexins in embryonic epithelial formation in Tribolium (van der Zee et al, revised at Development) and Drosophila (Giuliani et al, 2013a).

We have also elucidated the role of E-cadherin extracellular cleavage in apical extrusion, as one of the first step of tumor formation in mammalian epithelial cells (Grieve and Rabouille, 2014).

Unconventional secretion and mechanical stress For the last 6 years, we have aimed to understand how secretion and the secretory pathway respond to environmental stressful conditions. We have shown that dGRASP, a peripheral Golgi protein, is required to mediate Golgi bypass of integrins in the Drosophila follicular epithelium (Schotman et al, 2008). This pathway is part of the so-called “unconventional secretion pathway (Nickel and Rabouille, 2009; Nickel et al, 2012). This work is being pursued in collaboration with Erika Geisbrecht (Kansas University) in the Drosophila muscle (Wang et al, revised at Development). We showed that this pathway is regulated by dgrasp mRNA upregulation, targeting and localized translation, in response to mechanical stress (Schotman et al, 2009). We also showed that upregulated dgrasp mRNA is protected during targeting by the RNA protein HOW of the Quaking family (Giuliani et al, 2013b). To investigate whether this was also true in mammals, we made a knockout mouse for one of the two mammalian GRASP genes (GRASP65). However, the mouse is viable and does not have a phenotype (Veenendaal et al, 2014). We are planning to cross it to the KO of the second GRASP (GRASP55) that is available.

Secretion under nutrient stress Recently, we have focused our effort to understand how the functional organization of the secretory pathway responds to starvation and what is the signaling involved. We performed a targeted RNAi screen to identify kinases involved in the organization of the early secretory pathway and identified ERK7 as a key kinase in the response of Sec16 to serum starvation (Zacharogianni et al, 2011). We then investigated the response of the secretory pathway to amino-acid starvation and shows that it leads to the formation of a novel stress assembly with features of liquid droplets, the Sec bodies that are critical for cell vialibity during stress (Zacharogianni et al, revised). We then showed a role for ERES components (including Sec16 and the COPII subunits) in mediating protein translation inhibition (Zacharogianni et al, in preparation). In collaboration with Ernst Hafen and Hugo Stocker (ETH, Zurich, Switzerland), first to show a role for Rictor in heat shock induced stress granule formation (Zacharogianni et al, in preparation)

Future research

Nutrient stress, Sec bodies and cell response We plan to expand our research line to understand the nutrient stress cell response in mammalian cells and tumors. We also want to focus on understanding this response in vivo in Drosophila. Conversely, we want to investigate the molecular factors that drive Sec body formation, including the post-translational modifications of key factors (Sec24AB and Sec16) and the signaling pathways. We want to reconstitute Sec body formation in vitro or in a semi intact cell system and understand what is needed in the cytoplasm to allow phase transition driven by amino-acid starvation Considering the relevance of Sec body components in the formation of stress granules (that form in response to inhibition of protein translation), we want to understand how they play this role and why. In

- 92 - particular, we like to know whether specific mRNAs are protected. In this regard, we will investigate the translational and transcriptional response to amino-acid starvation. Both pathways are largely inhibited but some specific transcripts and proteins might be produced.

Mechanical stress and unconventional secretion of integrin subunits. There, we are planning to capitalize on the role of dGRASP in unconventional secretion of intergrins. This remains quite mysterious and possible tissue specific. We will also investigate if this happen in mammals by making a GRASP65/55 double knockout.

Societal relevance and societal impact (2008-2014).

As mentioned above, protein transport through the secretory pathway is a major anabolic ubiquitous pathway present in all eukaryotes. It is responsible for most of the cellular compartmentalization. It is used by almost all transmembrane proteins that define the function and identity of intracellular membrane organelles. As such, identification of molecular mechanisms underying its function and regulation is highly topical and as been rewarded by the Nobel prizes in Physiology and medicine 2013 to J. Rothman, R Schekmann and T. Sudhof. Furthermore, an increasing number of diseases and developmental defects are associated with mutations encoding proteins of the early secretory pathway (reviewed in Kondylis, Pizette and Rabouille, 2009).

Stress at the organismal level has a major impact on population’s health and well-being, whether heat, caloric, oxidative or mechanical, but it is still not completely understood. In particular, the response has two tenets, a systemic/hormonal one, and a cellular one that is thought to be autonomous. Here, we focus one the cellular response. During stress at the cellular level, anabolic pathways are generally shut down or stalled and most of the community effort has been concentrated on protein and ribosome synthesis (with focus on the mTORC1 pathway). In my group, we investigated the effect on stress on a different major anabolic, protein transport through the secretory pathway, and made a number of important discoveries.The first is that the cellular response to the stress of amino-acid starvation (with the formation of Sec bodies) is critical for cell survival, thus helps the cells to cope with stress but also contribute to their fitness upon re-addition of nutrient. Unexpectedly, this influences the protein translation response. As we are now planning to investigate whether Sec bodies form in solid tumors (the core of which is often “starved”, the modulation of their formation could have a strong effect on their growth. If so, as we have shown that GNC2 is required (at least partially) for Sec body formation, pharmacological inhibition of this kinase would also modulate the tumor growth.

We have also investigating protein transport through the secretory pathway under mechanical stress and found that the transport of integrins is modulated, at least in in two different tissues in Drosophila, via dGRASP. Indeed, instead of using the classical secretory pathway, some integrin subunits bypass the Golgi in a dGRASP dependent fashion. More and more substrates are now shown to be unconventionally secreted, and this provides an additional level in the regulation of protein function (in term of post-translational modifications). As such, this is complementary to understanding transcription and translation regulation. We do not understand completely the impact of these results. When we have the opportunity to pursue this project in a mammalian system, using double mutant GRASP65 (that we made) and GRASP55 mice (that is available), we will see whether this pathway operates outside Drosophila and whether it also modulates integrin delivery upon mechanical stress. As those molecules are key for cell adhesion and epithelium integrity, this pathway is potentially critical as tumor suppressor.

- 93 -

- 94 -

Catherine Robin

Key publications (2008-2014)

Robin C*, Bollerot K*, Mendes S*, Haak E, Crisan M, Cerisoli F, Lauw I, Kaimakis P, Jorna R, Vermeulen M, Kayser M, van der Linden R, Imanirad P, Verstegen M, Nawaz-Yousaf H, Papazian N, Steegers E, Cupedo T, and Dzierzak E. (2009) Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development. Cell Stem Cell 5: 385- 395.

Boisset J-C, Van Cappellen W, Andrieu-Soler C, Galjart N, Dzierzak E and Robin C. (2010) In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464: 116-120.

Boisset JC, Andrieu-Soler C, van Cappellen WA, Clapes T, and Robin C. (2011) Ex vivo time-lapse confocal imaging of the mouse embryo aorta. Nature protocols 6: 1792-1805.

Drabek K, Gutiérrez L, Vermeij M, Clapes T, Patel S, Boisset J-C, van Haren J, Pereira A, Liu Z, Akinci U, Nikolic T, van Ijcken W, van den Hout M, Meinders M, Melo C, Sambade C, Drabek D, Hendriks R, Philipsen S, Mommaas M, Grosveld F, Maiato H, Italiano J, Robin C, and Galjart N. (2012) The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance. Cell Reports 2: 781-788.

Boisset JC, Clapes T, Van Der Linden R, Dzierzak E, and Robin C. (2013) Integrin αIIb (CD41) plays a role in the maintenance of hematopoietic stem cell activity in the mouse embryonic aorta. Biology Open 2: 525-532.

- 95 - Dr. Catherine Robin Hematopoiesis and stem cells during embryonic development

Group members Postdocs: Jean-Charles Boisset (end: 01/01/14), Gaëlle Billoud (end: 01/05/14) Fanny Sage, Laurent Yvernogeau Graduate students: Jean-Charles Boisset (PhD defense: 25/10/12), Thomas Clapes (PhD defense: 17/09/14), Chloé Baron, Panagiota Giardoglou (start: 16/09/14), Anna Katharina Klaus (start: 16/10/14) Technicians Mary Stevens (end: 01/02/14), Hans Meijer

Curriculum vitae group leader

Name: Catherine Robin Date of birth: 20 January 1971 Nationality: French

Education/positions 1994-1995 MSc, Denis Diderot University (Paris VII) 1995-2000 PhD, INSERM U362 (Hematopoiesis and Stem Cells Lab), Villejuif Denis Diderot University. Supervisors: W. Vainchenker, L. Coulombel Thesis entitled “Identification of human hematopoietic stem cells by in vivo transplantation into NOD-SCID mice”. 2000-2006 Post-Doc, Erasmus University Medical Center, Cell Biology Dept., Rotterdam, in the lab of Prof. E. Dzierzak

2006-2008 Group leader, Erasmus University Medical Center, Dept. of Cell Biology, Rotterdam 2008-2010 Group leader, Erasmus University Medical Center, Erasmus Stem Cell Institute for Regenerative Medicine (ESI), Rotterdam 2010-2013 Assistant/Associate Professor, Erasmus University Medical Center, ESI, Rotterdam 2013-Present Group leader, Hubrecht Institute, Utrecht, the Netherlands; Cell Biology Dept., University Medical Center Utrecht

Memberships • International Society for Experimental Hematology (ISEH) • International Society for Stem Cell Research (ISSCR) • Dutch Society for Developmental Biology (DSDB) • AcademiaNet (database of excellent scientists)

Awards • European Marie Curie individual postdoctoral fellowship (HPMF-CT-2000-00871) • French postdoctoral fellowship “La ligue nationale contre le cancer” • Vereniging Trustfonds Erasmus MC Rotterdam (twice) • Keystone Symposia scholarship • ISSCR Travel award • Talent day Erasmus Universiteit Rotterdam Award (twice)

Other activities Co-organizer of “Introduction to Stem Cells Course” (open for the Regenerative Medicine Utrecht PhD Program and the Cancer, Genomics, Developmental Biology Master/PhD Programs) in Utrecht. Organizer of the ESI Erasmus international lecture series and ESI Erasmus literature meetings. Invited individual lecture: 2008 Paterson Institute for Cancer Research, University of Manchester (UK) Ludwig-Maximilians-Universität, Munich (DE) 2009 Institute for Physiological Chemistry/Pathobiochemistry, Muenster University (DE), Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford (UK). 2010 Hôpital Necker-Enfants Malades, INSERM U768, Paris (FR). 2012 EMBL Eminent Speaker Seminar Series, Monterotondo (IT)

- 96 - Ludwig-Maximilians Universität, Munich (DE) VUmc, Dept. Molecular cell biology & Immunology, Amsterdam (NL) 2014 “Centre de biologie du développement”, Paul Sabatier University, Toulouse (FR), Paterson Institute for Cancer Research, University of Manchester (UK).

Invited speaker on meetings (2008-2014) 2008 Stem Cells in Development and Disease (SCDD), Amsterdam (NL) 5th int. meeting cell therapy, bioengineering, regenerative medicine, Nancy (FR) 2009 BD-EMCR FlowDay, Rotterdam (NL) 38th ISEH Annual Scientific Meeting, Athens (GR) Stem Cells in Development and Disease (SCDD), Rotterdam (NL) 2010 Stem Cell Society Singapore Symposium (SCSS), Singapore (SG) Waddensymposium on Stem Cell Renewal, Texel (NL) Molecular Haemopoiesis 13, Institute of child health, London (UK) CHO “Club of Hematopoiesis and Oncogenesis”, Presqu’ile de Giens (FR) Traute Schroeder-Kurth Symposium, University of Wurzburg, Wurzburg (DE) International Society for Stem Cell Research, 8th Meeting (ISSCR), SF (USA) 2011 Inaugural Dutch Society for Developmental Biology (DSDB), Utrecht (NL) UPMC Stem Cell Initiative, Paris (FR) 2012 EMBL Eminent Speaker Seminar Series, Monterotondo (IT) 2nd Trippenhuis meeting, Dynamic interactions in cell biology, Amsterdam (NL) 2013 2nd Igakuken International Symposium on HSC development, Tokyo (JP) Spetses Meeting (Summer school 2013), Island of Spetses (GR) 2014 48th meeting of European society for clinical investigation (ESCI), Utrecht (NL) International conference on leukocyte trafficking (SFB914), Munich (DE) CHO “Club of Hematopoiesis and Oncogenesis”, Grasse (FR)

Grants (2008-2014) 2007-2011 Vidi Dutch young investigator grant (NWO) (917-76-345) Principal investigator: Catherine Robin € 600,000 “In vivo real time imaging of hematopoietic stem cell development” 2010-2014 Landsteiner Stichting voor Bloedtransfusie Research grant (LSBR 1025) Co-Principal investigator: Catherine Robin € 388,000 “Role of the microtubule network in the generation and expansion of the first hematopoietic stem cells” 2013-2018 ERC consolidator grant (220-H75001 EU/ HSCOrigin – 309361). Principal investigator: Catherine Robin €1,500,000 “From mesoderm to hematopoietic stem cell commitment: cellular and molecular events occurring during mouse embryonic development” 2014-2016 Utrecht university life sciences seed award Co-Principal investigator: Catherine Robin € 100,000 “An ex vivo three-dimensional bio-reconstituted bone marrow niche to study normal hematopoiesis and bone-residing malignancies”

A postdoc fellow (L. Yvernogeau) in the lab has been awarded an EMBO short-term fellowship.

Patents etc. (2008-2014) None

Previous research

Hematopoietic stem cells (HSCs) are self-renewing multipotent cells that produce all blood cell types during the entire life of an organism. Hence, they are the only cell type that can be used to replenish the bone marrow (BM) in patients with blood-related disorders. One major challenge in the field of stem cell research is to generate large quantities of these very rare cells in vitro for research and clinical use. This is extremely difficult because not all steps leading to HSC generation in vivo have been elucidated yet. The overall research goal of my lab is to elucidate the cellular and molecular events leading to HSC production and expansion as they occur in vivo during embryonic development.

- 97 - We notably focus on understanding the developmental origin(s) of HSCs, the mechanistic events underlying HSC generation, the composition and function of the different HSC supportive microenvironments (or niches) during ontogeny, and the cellular and molecular signature of HSCs and HSC precursors. These processes are largely unclear and we have made substantial progress over the past years.

During my doctoral research period in the laboratory of Dr. Vainchenker in France, my research focused on human hematopoiesis. I notably identified at clonal level HSCs in human cord blood, adult BM and embryos (in collaboration with Dr. Péault) by developing and using in vitro clonal cultures and NOD-SCID xenotransplantation assays (Robin et al., 1999; Tavian et al., 2001). I subsequently joined Prof. Dzierzak’s laboratory in the Cell Biology Department of Prof. Grosveld in the Netherlands to expand my knowledge in mouse developmental hematopoiesis. There I studied the first HSCs generated at embryonic day (E)10.5 of mouse development in the intraembryonic aorta-gonad- mesonephros (AGM) region. Using a transgenic mouse model (Ly6A-GFP; expressing GFP under the control of Ly6A, which codes for Sca-1), we demonstrated that all HSCs are restricted to the GFP+ fraction during embryonic development and in adults (Ma et al., 2002). These cells are specifically localized in clusters of cells (intra-aortic hematopoietic clusters or IAHCs, where HSCs reside) attached to the aortic endothelium and also within the endothelial layer lining the wall of the dorsal aorta, strongly suggesting a vascular endothelial origin of the first HSCs (De Bruijn et al., 2002). We also identified signaling molecules involve in the survival and expansion of HSCs in the AGM region, including IL-3, BMP-4 and two BMP antagonists (Gremlin and Noggin) (Robin et al., 2006; Durand et al., 2006, 2007; Robin et al., 2010). Although I continued to study human hematopoiesis (Robin et al., 2009), I mainly used the mouse model during my postdoc research period.

After my training in adult and embryonic human/mouse hematopoiesis, I build up my own line of research as an independent researcher (in 2006). We started off by developing an original approach to directly observe in real-time the formation of the first IAHCs (and therefore HSCs) in the aorta of the mouse embryo. Most of the research studies performed to investigate the hematopoietic embryonic development were based on the analysis of fixed cells/tissues dissected at a particular time point of development. Although these studies were very informative, they remained static and excluded the possibility to observe the lineage relationship between different cell types, as well as their interaction and migration in their tissue of origin. Studies were also based on lineage tracing experiments, which are retrospective and analyze the progeny of HSCs rather than HSCs directly. It was thus important to observe these cells in their living physiological environment, directly in the embryo. Although it was more or less accepted that HSCs derive from specialized endothelial cells named “hemogenic”, whether the hemogenic endothelial cell to HSC transition (EHT) actually occurs in the mouse embryonic aorta (and/or in other sites such as the yolk sac or placenta where HSCs are also found) was unclear. In contrast to previous studies, we developed an experimental approach where we combined confocal live imaging and new dissection procedures, to visualize in real-time HSC generation directly in the native context of the living aorta. Live studies in mouse embryos have thus far been hampered because the aorta is located deeply within the embryo and the embryo itself is opaque. We developed a new procedure to access and visualize live cells in the aorta of non-fixed Ly6A-GFP embryos or CD41-YFP embryos (CD41 is considered as the first marker indicative of hematopoietic lineage commitment). The embryos were dissected at E10.5 (time of HSC detection as demonstrated by long-term transplantation experiments). Fluorochrome-conjugated antibodies (against CD31, an endothelial marker) were injected directly inside the aorta to specifically stain the endothelium. Embryos were then cut into thick transverse slices to optically access the aorta. Embryo slices were subsequently cultured directly under the confocal microscope and time-lapse imaged for up to 15 h (Boisset et al., 2011). In these conditions we imaged, for the first time, the dynamic de novo emergence of phenotypically defined HSCs (CD31+, Ly6A+, CD41+) directly from the ventral aortic endothelium (Boisset et al., 2010). Staining of the embryo slices after imaging proved that the emerging cells also expressed the HSC marker c-kit. Our findings definitively proved that HSCs are generated inside the aorta from hemogenic endothelial cells. Our conclusions were supported at the time by three other studies performed on zebrafish embryos. Our work was recognized as a major breakthrough in the field of hematopoiesis and stem cell biology and underlined by an editorial in Nature and research highlights (Nature Reviews Molecular Cell Biology (11, 2010), Cell Stem Cell (6:289-90, 2010)). Because our new experimental approach was of general interest for different research fields, our technique was also published in Nature Protocols (Boisset et al., 2011). Now that we have developed a system to follow the dynamic emergence of HSCs in the live mouse embryo aorta, it will allow us to elucidate each steps involved in EHT. This process is far from being

- 98 - clear in the mouse embryo. It will also help to identify the defective mechanisms in hematopoietic mutants (e.g. in Runx1-/-, which lack both IAHCs and HSCs).

We also focused on a better phenotypic characterization of HSCs throughout mouse development, which is important to localize and isolate these rare cells. We focused on the expression and function of integrins during mouse HSC development. CD41 (αIIb), a membrane glycoprotein, associates with CD61 (β3) to form the integrin αIIbβ3. CD61 can also bind CD51 (αv) to form the integrin αvβ3. CD41 is most likely the first marker indicative of hematopoietic lineage commitment. However, the expression pattern and functional role of these integrins on HSCs throughout mouse embryonic development were still unclear. We found that all HSCs from the dorsal aorta express intermediate (int) levels of both αIIbβ3 and αvβ3 integrins. In comparison, all HSCs found later on in the placenta only express αvβ3 integrins while most fetal liver HSCs do not. Thus, CD41 expression on HSCs is downregulated throughout development. This downregulation is not tissue but time specific during embryonic development because CD41 starts to be downregulated on HSCs in the aorta at E12 (when HSC activity switches to other sites). Thus, CD41 is expressed only on the newly generated HSCs (Robin et al., 2011). αIIbβ3 integrin is an exquisite marker of AGM HSCs and allows a massive enrichment when combined with other markers (e.g. CD41intCD61intCD45intckit+). Integrin expression can thus be used to discriminate and isolate HSCs during mouse embryonic development. Finally to determine whether CD41 plays a functional role in the production of the first HSCs, we tested the HSC activity in AGMs dissected from embryos knock-out for CD41. After transplantation into irradiated adult recipients, we observed a dramatic decrease of HSC activity when the mice where transplanted with CD41-/- cells compared to wild-type cells (Boisset et al., 2013). Together our results point to an essential role of the integrin αIIbβ3 (CD41/CD61). CD41 is not only a reliable marker for AGM HSCs but it also plays a major role to maintain HSC activity in the AGM. Whether the role of αIIbβ3 is to properly anchor HSCs to the aortic endothelium at the time of their emergence until they are fully competent to colonize the fetal liver or to allow proper signaling pathways remain unclear to date.

Future research

We will continue to develop state-of-the-art technology to understand all steps leading to HSC formation and also muscle stem (satellite) cell formation (a new stem cell research line in the lab). We mainly use the mouse and chicken models (and will use the zebrafish model when required).

Our future research plans will focus on the following issues:

1- Biomechanical events involved in EHT and consecutive production/expansion of HSCs during embryonic development.

We observed intense cellular movements during EHT, highlighting important roles for cell adhesion molecules and microtubule (MT) cytoskeleton rearrangement. To better understand the basic biomechanics of embryonic HSC formation, we focused on the MT-stabilizing factors, CLASPs (Cytoplasmic Linker Proteins associated proteins). We have previously shown that adult Clasp2-/- mice have a drastic HSC diminution in the BM showing that CLASP2-mediated MT stabilization is required for adult HSC maintenance (Drabek et al., 2012) (Collaboration: Dr. N. Galjart, Rotterdam). Recent data have shown that the defect starts as soon as HSCs enter the BM, with progressive loss of c-kit expression on HSC surface. We will further study: • How CLASP2 is involved in the regulation of c-kit expression in HSCs. • Whether CLASP2 already plays a role during embryonic HSC production. • The possible collaboration of CLASP2 with integrins.

2- Function of IAHCs and molecular program leading to hemogenic endothelium, IAHC and HSC production.

We recently found that IAHCs mainly contain HSC precursors (pre-HSCs), i.e. capable of long-term multilineage reconstitution in newborns but not in adults (Boisset et al., in revision). Successful secondary transplantations proved that IAHCs could mature into HSCs in vivo. Therefore, IAHC pre- HSCs are an intermediate between hemogenic endothelium and HSCs. We will further study: • Whether IAHCs produce HSCs in vivo via maturation after migration to the fetal liver (main HSC site at mid-gestation). To elucidate at the molecular level the successive steps leading to IAHC and HSC formation, we will:

- 99 - • Perform RNA-Sequencing on single IAHCs (isolated by mechanical pick up), single IAHC cells, HSCs and hemogenic/non-hemogenic endothelial cells, isolated before and after HSC detection (Collaboration: Prof. van Oudenaarden). • Study the signaling pathways and transcription factors expressed during HSC commitment. • Manipulate the most promising pathways and test the role of candidate genes (e.g. conditional knock-out mice, CRISPR/Talens technology in zebrafish embryos, RNAi).

The same research questions are also examined in the chicken embryo model.

3- Anatomical origin of HSCs during embryonic development and yolk sac blood island formation.

The anatomical site of HSC origin remains controversial in mammals because the blood circulates at the time of HSC detection. We are currently developing a novel in vivo embryo rescue assay by: • Isolating cells from different tissues (yolk sac, allantois, caudal part of E8 embryo) before the circulation starts. • Transplanting the cells in utero under ultra-sound guidance directly into E9 hematopoietic mutant embryos (known to die at birth due to HSC defect). Embryos will be rescued only if the transplanted cells can generate potent HSCs. We already have the proof of principal that the injection technique works with E13 fetal liver sorted HSCs that could provide long-term multilineage hematopoietic reconstitution in the transplanted developing embryos at adult stage.

The anatomical origin of HSCs will also be examined in the chicken embryo model by: • Isolating cells from yolk sac, allantois, IAHCs, para-aortic hematopoietic foci (PAF, equivalent of mammalian fetal liver site) and pre-somitic mesoderm tissue. • Establishing a transplantation/grafting assay in the chicken embryo (underneath the aorta in the PAF region). • Studying long-term hematopoietic production in the transplanted developing embryos at adult stage.

We will also analyze the formation of the mouse yolk sac blood islands to determine whether the first hematopoietic cells are generated by hemangioblasts and/or hemogenic endothelium (i.e. by time- lapse live confocal imaging of (crossed) fluorescent mouse lines including CD41-YFP, Flk1-IRFP (to be generated), Brachyury-GFP, Prox-Runx1-RFP and Dis-Runx1-GFP) (Collaboration: Dr. Lacaud, Manchester).

4- Reconstruction of embryonic niches in 3D-scaffolds to promote HSC generation and expansion in vivo.

The intrinsic and extrinsic events dictating the hemogenic fate in endothelial cells and the consequent HSC production are largely unknown and therefore extremely difficult to reproduce in vitro. We will: • Use the RNA-Sequencing data (comparing hemogenic/non-hemogenic endothelial cells; see point 2) to induce a hemogenic potential in mouse adult endothelial cells (Collaboration: Dr. Geijsen). • Develop a novel approach where embryonic microenvironments will be reconstructed in vivo to promote the ability of hemogenic induced endothelial cells to produce/expand functional HSCs. In preliminary experiments, the microenvironment of dissected embryonic aorta and fetal liver will be seeded separately in 3D scaffolds, thereafter implanted in vivo under the skin of immunodeficient mice (Collaboration: Dr. Martens, Amsterdam). Our approach should provide in vivo the two complete and complex microenvironments needed during development for HSC production and expansion, respectively. Our study will help to identify the important compounds/factors of the supportive HSC niches with the possibility to manipulate them in vivo.

We will also use 3D bioprinting technology (available at UMC, Utrecht) to reconstruct embryonic and also BM microenvironments (e.g. to investigate normal physiological interactions/processes occurring between HSCs and their complex microenvironment). The ex vivo BM niche will be developed to create expansion methodologies for human HSCs and also to study the role of the BM niche in the pathogenesis of diseases (Collaboration: Dr. Alblas/Dhert lab, UMC).

5- Contribution of pre-somitic mesoderm to different lineages.

- 100 - We will study all derivatives from the pre-somitic mesoderm (PSM) during embryonic development and in adults with a particular focus on (hemogenic) endothelium, HSCs and muscle satellite cell potential contribution (related to points 3 and 6). We will: • Construct a mouse line to specifically label PSM-derived cells (i.e. Tamoxifen inducible Tbx6 (enhancer)-CRE mouse line, further crossed with Rosa-YFP or Confetti mouse lines). • Trace Tbx6+ cells and progeny during embryonic development and adult stage.

6- Temporal emergence of the satellite cell potential during mouse embryonic development and adult muscle formation.

We recently started to investigate muscle stem (satellite) cell formation during embryonic development and adults. We will study: • The precise developmental time point when satellite cell activity starts (by transplanting injured NOD-SCID mice with Pax7-GFP+ cells isolated at different embryonic developmental points from anatomical structures known to contain embryonic myogenic precursors, and analyzing the contribution to fiber regeneration) (Collaboration: Dr. Tajbakhsh, Paris). • The molecular events leading to the emergence of satellite cell potential (by single cell RNA- Sequencing on Pax7-GFP+ cells sorted before and after stem cell activity detection (Collaboration: Prof. van Oudenaarden). • Myofiber formation during development and upon regeneration by using a lineage tracing approach (by generating a Pax7ERCre/Confetti mouse line) (Collaboration: Prof. Clevers).

Societal relevance and societal impact (2008-2014)

My projects are fundamental in nature but have also societal relevance. They mainly aim to answer biological long-standing questions about HSC production and regulation during embryonic development. We also focused on a better phenotypic characterization of HSCs throughout mouse development, which is important to localize and isolate these cells for further molecular analysis. These research topics are important, knowing the crucial need of an unlimited source of HSCs for research and clinical use (to treat blood related diseases). Knowing the intrinsic and extrinsic regulators leading to stem cell production in vivo will help to design in vitro/ex vivo HSC culture conditions. A better knowledge of normal HSC production is also essential to understand HSC dysregulation. Such knowledge is also relevant for other stem cell fields since key pathways and regulatory factors are most likely highly conserved.

The origin of HSCs has been the subject of intense debate over the past years. HSCs were proposed to derive either from specialized endothelial cells (named “hemogenic”) or from the mesenchyme, and to originate either from extra- (yolk sac, placenta) or intra-embryonic (aorta) sites. I focused my research over the past years on these long-standing and controversial questions. The opaqueness of the mouse embryo and the deep location of the dorsal aorta have severely hampered direct imaging. We overcame this difficulty by developing an embryo dissection procedure to prepare thick transversal embryo slices of E10.5 Ly6A-GFP transgenic mouse embryos (whereby the HSC marker Ly6A-GFP marks all HSCs and a subset of CD31+ cells in the endothelial cell wall). This approach allows optical access of the inside of the living mouse aorta. We then developed a new staining method by injecting directly labeled antibodies (classically used for cell suspension flow cytometry analysis) inside the aorta to label the endothelium (CD31+). Embryo slices were then imaged by time-lapse confocal imaging. We witnessed round Ly6A-GFP+CD31+ cells budding from the aortic wall into the lumen. The hematopoietic nature of these emerging cells was confirmed by c-kit and CD41 hematopoietic marker expression and by the absence of such events in Runx1 null embryos. Thus, we were the first to image and therefore to definitively prove that HSCs originate directly from the hemogenic endothelium on the ventral side of the dorsal aorta. This mechanism is conserved among different species since our data were confirmed by three other research groups who also observed HSC production directly from the aortic endothelium of the zebrafish embryo. These data, and future descriptions of the HSC developmental pathways, are very important and should help to generate large quantities of HSCs (e.g. from pluripotent progenitors) for therapeutic purposes. Getting an easier and safer supply of HSCs for transplantation protocols is indeed of particular interest for clinicians and consequently for patients. Indeed, the difficulty to find compatible donors and the risk of graft-versus-host disease (associated to high morbidity and mortality in allogeneic HSC transplantation) are major concerns. The production of HSCs from new sources is of great interest. We and others have now demonstrated that all HSCs derive from hemogenic endothelial cells.

- 101 - Therefore, generating HSCs from vessel cells by inducing a hemogenic potential in endothelial cells collected via non-invasive procedures, from adult donor/patient circulating blood or usually discarded neonatal tissues (e.g. umbilical cord, source that might even produce more potent HSCs due to the immature stage of the endothelial cells), is a very attractive new approach to generate HSCs. It would have a tremendous impact in clinic.

Our work was recognized as a major breakthrough in the field of hematopoiesis and stem cell biology (Boisset et al., Nature). It was largely advertised over the news in the Netherlands. It was broadcasted on the head news of the first Dutch national TV (NOS 1), Dutch local TV (Rijnmond TV) and by several national newspapers (Trouw, NRC, Volkskrant, nu, AD) and scientific journals in France (La Recherche, Médecine/Sciences). There was also an editorial in Nature and research highlights notably in Nature Reviews Molecular Cell Biology (11, 2010) and Cell Stem Cell (6:289-90, 2010). Because our new experimental approach was of general interest for different research fields, our technique was published in Nature Protocols (2011). Since, our time-lapse confocal imaging on embryo slices has been distributed to other lab involved in stem cell field or other. I have also collaborations in which my lab contributes via key developmental hematopoiesis and stem cell intellectual and technical expertise.

An other interesting and exciting aspect of my research is that it has been accessible to a larger public since several images and movies generated in my group have been exposed, at the symposium "Beauty and Science" which was held for 5 months at the Museum Boymans van Beuningen in Rotterdam (The Netherlands) and in the museum of natural history in Shanghai (China). One of our movies has also been selected and exposed at the exhibition “Rollercoaster, The Image in the 21st Century” at the MOTI museum of Breda (The Netherlands).

- 102 -

Alexander van Oudenaarden

Key publications (2008-2014)

Junker JP, Noël ES, Guryev V, Peterson KA, Shah G, Huisken J, McMahon AP, Berezikov E, Bakkers J and van Oudenaarden, A (2014) Genome-wide RNA tomography in the zebrafish embryo, Cell, in press.

Ji N, Middelkoop TC, Mentink RA, Betist MC, Tonegawa S, Mooijman D, Korswagen HC and van Oudenaarden A. (2013) Feedback control of gene expression variability in the Caenorhabditis elegans Wnt pathway, Cell 155(4):869-80.

Neuert G, Munsky B, Tan RZ, Teytelman L, Khammash M and van Oudenaarden A. (2013) Systematic identification of signal-activated stochastic gene regulation, Science 339(6119):584-7.

Itzkovitz S, Blat IC, Jacks T, Clevers H and van Oudenaarden A. (2012) Optimality in the development of intestinal crypts, Cell 148(3):608-19.

Raj A, Rifkin SA, Andersen E and van Oudenaarden A. (2010) Variability in gene expression underlies incomplete penetrance, Nature 463(7283):913-8.

- 103 - Prof. Dr. ir. Alexander van Oudenaarden Quantitative biology of development & stem cells

Group members Postdocs: Jean-Charles Boisset, Magda Bienko, Nicola Crosetto, Siddharth Dey, Dominic Grün, Jan-Philipp Junker, Anna van Oudenaarden-Lyubimova, Stefan Semrau, Nikolai Slavov. Graduate students: Susanne van den Brink, Lennart Kester, Dylan Mooijman, Mauro Muraro Gurrachaga, Abel Vertesy, Kay Wiebrands. Technicians: Bastiaan Spanjaard

Curriculum vitae group leader

Name: Alexander van Oudenaarden Date of birth: 19 March 1970 Nationality: Dutch

Education/positions 1988 – 1993 MSc Materials Science and Engineering 1988 – 1993 MSc Physics 1994 – 1998 PhD Physics 1998 – 1999 Postdoctoral research, Department of Chemistry, Stanford University, Stanford, CA, USA, Laboratory of Prof. S.G. Boxer, Micropatterning of supported phospholipid bilayers 1998 – 1999 Postdoctoral research, Department of Biochemistry, Stanford, CA, USA, Laboratory of Prof. J.A. Theriot, Force generation of polymerizing actin filaments 2000 – 2004 Assistant Professor of Physics, Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA. 2004 – 2008 Associate Professor of Physics with tenure, Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA. 2008 – 2009 Visiting Professor, Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands. 2008 – 2014 Professor of Physics, Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA. 2009 – 2014 Professor of Biology, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. 2009 – 2014 Extramural faculty member of the Koch Institute for Integrative, Cancer Research at MIT, Cambridge, MA, USA. 2012 – present Director Hubrecht Institute 2013 – present Professor, Faculty of Science, Utrecht University, Utrecht, The Netherlands. 2013 – present Professor, University Medical Center Utrecht, Utrecht, The Netherlands.

Memberships 2014 – present Member of the Royal Netherlands Academy of Arts and Sciences (KNAW) 2014 – present Advisory editorial board member Molecular Systems Biology 2013 – present Member of the Scientific Advisory Board of the European Molecular Biology Laboratory (EMBL) 2012 – present Member of the Scientific Advisory Board of the Max Planck Institutes for Molecular Cell Biology and Genetics 2011 – present Member of the Scientific Advisory Board of the Whitehead Institute for Biomedical Research

Awards 2012 ERC Advanced Investigator Award 2012 Dutch Organization for Scientific Research (NWO) Vici Award 2008 NIH Director’s Pioneer Award 2008 Guggenheim Fellow 2007 School of Science Prize for Excellence in Graduate Teaching

- 104 - 2001 Keck Career Development Professor in Biomedical Engineering 2001 Alfred Sloan Research Fellow 2001 NSF CAREER award 2000 Edgerly Science Partnership Award 1998 Andries Miedema Award for best PhD-research in the field of condensed matter physics in the Netherlands, awarded every other year by Fundamental Research on Matter (FOM). 1998 Dutch Organization for Scientific Research (NWO) TALENT stipendium. 1998 PhD Applied Physics, cum laude. 1994 Award for best undergraduate research in Materials Science, yearly award by Delft University of Technology. 1993 M.S. Materials Science and Engineering, cum laude.

Other activities 10/2009 – 07/2012 Director of the MIT Center for Single-Cell Dynamics in Cancer (NIH/NCI funded U54 Physical Sciences-Oncology Center). The goal of this center is use both theoretical and experimental approaches inspired by Physics to attack important problems in cancer biology by developing novel technology and analytical/computational methods to track the dynamics of cancer at the single cell level. 06/2007 – 12/2011 Organizer of CSB (Computational and Systems Biology) seminar series. 01/2005 – 12/2007 Associate Editor Biophysical Journal 06/2004 – 07/2006 Course Faculty at the Marine Biology Laboratory (Woods Hole) Summer Course ‘Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches’ 09/2002 – 12/2009 Lecturer and creator of MIT Graduate course 7.81/8.591/9.531 Systems Biology. This course is offered annually during the Fall semester. The course provides an introduction to the mathematical tools that are used to dynamically model gene and protein networks. The course is attended by about 60-70 Graduate students (about 50% having a background in biological sciences and 50% having a background in physical sciences). This course was awarded with the School of Science Prize for Excellence in Graduate Teaching.

Thesis advisor for 2008 John Tsang (Harvard) 2008 Carlos Gomez-Uribe (MIT) 2009 Dale Muzzey (Harvard) 2009 Qiong Yang (MIT) 2010 Shankar Mukherji (MIT) 2010 Bernardo Pando (MIT) 2011 Hyun Youk (MIT) 2011 Mei Lyn Ong (MIT) 2011 Rui Zhen Tan (MIT) 2012 Miaoqing Fang (MIT) 2012 Dong hyun Kim (MIT) 2012 Jialing Li (MIT) 2013 Annalisa Pawlosky (MIT) 2013 Ni Ji (MIT) 2014 Yannan Zheng (MIT) 2014 Clinton Hansen (Harvard) 2014 Sandy Klemm (MIT) 2014 Ya Lin (Harvard) 2014 Apratim Sahay (MIT)

Keynotes 25/09/2014 EMBO workshop ''Unraveling Biological Secrets by Single-cell Expression Profiling", Heidelberg, Germany. 29/04/2014 Annual VIB (Flemish Institute for Biotechnology) Conference, Blankenberge, Belgium. 04/10/2013 Haldane Lecture at John Innes Centre, Norwich, UK. 18/06/2013 SignGene consortium, Berlin, Germany. 31/10/2013 International Conference on Systems Biology (ICSB) 2013, Copenhagen, Denmark.

- 105 - 16/04/2013 Netherlands Bioinformatics Conference 2013, Lunteren, The Netherlands. 24/10/2012 10th Dutch Chromatin Meeting, University of Amsterdam, Amsterdam, The Netherlands. 28/08/2011 International Conference on Systems Biology (ICSB) 2011, Mannheim, Germany. 10/03/2011 The Dutch Annual Biophysical Meeting, Veldhoven, The Netherlands. 17/10/2010 Stochasticity in Cell and Developmental Processes, Company of Biologists, Windsor, UK. 26/09/2010 Whitehead Institute Retreat, Waterville Valley, NH, USA.

Other invited talks 12/12/2014 “Marie Curie Seminar”, Curie Institute, Paris, France. 05/11/2014 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. 14/10/2014 Chromatin and Epigenetics: From Omics to Single Cells, IGBMC Strasbourg, France. 08/09/2014 Single-cell Genomics 2014, Stockholm, Sweden. 17/06/2014 International Society for Stem Cell Research (ISSCR) 2014, Vancouver, Canada. 06/06/2014 Symposium TUe-UMCU alliance, Utrecht, The Netherlands. 16/04/2014 Seminar Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. 15/04/2014 D-BSSE seminar, ETH-Zurich, Basel, Switzerland. 05/04/2014 NanoFront Kick-off event, Delft, The Netherlands. 18/03/2014 Utrecht M. Sc. Student seminar, Utrecht, The Netherlands. 11/03/2014 Philips Centennial Lecture, Philips Research, Eindhoven, The Netherlands. 05/03/2014 Launch of the UMC Center for Molecular Medicine, UMC Utrecht, The Netherlands. 27/02/2014 van Leeuwenhoek Lectures on BioScience, Leiden University, The Netherlands. 11/02/2014 Frontiers in Science in the Low Countries, Erasmus University, Rotterdam, The Netherlands. 29/01/2014 Debeye Colloquium, Utrecht University, Utrecht, The Netherlands. 22/01/2014 Physics@FOM, Veldhoven, The Netherlands. 10/01/2014 Cancer Genomics Netherlands meeting, Utrecht, The Netherlands. 06/12/2013 IBL Symposium 2013 ‘Biological networks: from molecules to society’, Leiden University, The Netherlands. 25/11/2013 Systems Biology@NL 2013, Egmond aan Zee, The Netherlands. 11/11/2013 NCMLS Symposium, Nijmegen, The Netherlands. 11/10/2013 Abcam Meeting on Single-cell Analyses in Stem Cell Research, Munchen, Germany. 02/10/2013 Single-cell Genomics 2013, Weizman Institute, Rehovot, Israel. 22/09/2013 The 5th EMBO meeting, Amsterdam, The Netherlands. 06/09/2013 NKI (Dutch Cancer Institute) seminar series, Amsterdam, The Netherlands. 08/07/2013 The Physical Biology of Stem Cells Conference, Cambridge, UK. 27/06/2013 Dutch Systems Biology Conference, Wageningen University, The Netherlands. 22/04/2013 25th Anniversary Symposium of the Bijvoet Center for Biomolecular Research, Utrecht, The Netherlands. 08/04/2013 AMOLF colloquium, Amsterdam, The Netherlands. 07/03/2013 CSHL Single-cell Analyses Conference, Cold Spring Harbor, USA. 05/03/2013 1st Weizmann Student Symposium on Systems Biology, Weizman Institute, Rehovot, Israel. 26/02/2013 Gurdon Institute Seminar, Cambridge, UK. 15/11/2012 CGC meeting at the Royal Tropical Institute, Amsterdam, The Netherlands. 27/09/2012 CGDB seminar, Utrecht, The Netherlands. 24/09/2012 Quantitative Biology Symposium, Groningen, The Netherlands. 18/09/2012 Hydra VIII Summer School in Stem Cells and Regenerative Medicine. Hydra, Greece. 23/08/2012 Presidential Symposium, Annual Meeting of the International Society of Experimental Hematology, Amsterdam, The Netherlands. 18/07/2012 4th International Conference on Stem Cells and Tissue Formation, Dresden, Germany. 26/03/2012 Systems Biology Seminar Series, Duke University, Durham, NC, USA. 02/03/2012 American Physical Society (APS) Annual Meeting, Boston, MA 27/06/2011 Gordon Conference on ‘Cell Growth’, Biddeford, ME, USA. 23/06/2011 CSHL The future of biomarker discovery, Banbury, NY, USA. 26/05/2011 UCSF – Systems Biology Seminar Series, UCSF, San Francisco, CA, USA. 25/05/2011 UCSF – Stem Cell Seminar Series, UCSF, San Francisco, CA, USA. 28/04/2011 Systems Biology Seminar, UMass 09/02/2011 Systems Genetics Seminars, Harvard Medical School, Boston, MA, USA.

- 106 - 23/01/2011 Gordon conference on ‘Stochastic Physics in Biology’, Ventura, CA, USA. 20/01/2011 Biology Seminar, Columbia Univeristy, New York, NY, USA. 09/12/2010 Master class, ‘Cancer Genomics & Developmental Biology’, Utrecht, The Netherlands. 24/06/2010 Broad Institute Seminar Series, Cambridge, MA, USA. 17/04/2010 NSF workshop on cellular decision-making, Arlington, VA, USA 01/04/2010 UCSD Molecular Biophysics Seminar, UCSD, San Diego, CA, USA. 31/03/2010 Frontiers in Biology Seminar Series, Stanford, CA, USA. 23/03/2010 CSHL Systems Biology, Cold Spring Harbor, NY, USA. 11/03/2010 “Imaging transcription in living cells: A systems and computational approach”, Janelia Farm, Ashburn, VA, USA. 16/10/2009 Inaugural Symposium for The Sackler Institute for Biological, Physical and Engineering Sciences, Yale University, New Haven, CT, USA. 24/09/2009 NIH Pioneer Award Symposium, NIH, Bethesda, MD, USA. 02/09/2009 International Conference on Systems Biology (ICSB) 2009, Stanford, CA, USA. 28/08/2009 CSHL Mechanisms of Eukaryotic Transcription, Cold Spring Harbor, NY, USA. 20/04/2009 Systems Biology Symposium 2009. Institute for Systems Biology, Seatlle, WA, USA. 13/04/2009 Princeton Quantitative Biology Seminar Series, Princeton, NJ, USA. 27/03/2009 CSHL Computational Cell Biology, Cold Spring Harbor, NY, USA. 24/03/2009 Berkeley Systems Biology Seminars, University of California, Berkeley, CA, USA. 23/03/2009 Biophysics Lecture Series, Caltech, Pasadena, CA, USA. 01/03/2009 Annual Biophysical Society Meeting, Boston, MA, USA. 19/01/2009 AMOLF Seminar, AMOLF, Amsterdam, The Netherlands. 18/12/2008 Conference on Stochastic Gene Expression, AMOLF, Amsterdam, The Netherlands. 07/11/2008 Synthetic Biology Conference, Groningen, The Netherlands. 06/11/2008 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. 01/10/2008 Physics Seminar, Leiden University, Leiden, The Netherlands. 15/09/2008 EMBO/ESF BACNET 2008 Conference, Sant Feliu de Guixols, Spain. 20/06/2008 Dutch Cancer Institute (NKI) Seminar, Amsterdam, The Netherlands. 14/04/2008 Johns Hopkins University Biophysics Seminar, Baltimore, MD, USA. 05/04/2008 Experimental Biology Conference 2008, San Diego, CA, USA. 25/03/2008 Batsheva Seminar on Information Processing in Living Cells, En-Gedi, Israel. 23/01/2008 University of Connecticut, Health Center, Farmington, CT, USA.

Grants (2008-2014) 2013 – 2017 European Research Council (ERC) Advanced Grant € 2,500,000 2013 – 2017 Netherlands Organization of Scientific Research (NWO) Vici Award € 1,500,000 2013 – 2017 Netherlands Organization of Scientific Research (NWO) Gravity Program € 400,000 2013 – 2017 Skolkovo Stem Cell Institute € 2,000,000 2013 – 2015 NIH R01 grant € 600,000 2009 – 2013 NIH/NCI - The MIT Center for Single-Cell Dynamics in Cancer € 1,000,000 2008 – 2012 NIH Director’s Pioneer Award $ 2,500,000 2008 – 2012 NSF Collaborative Research Grant $ 400,000 2003 – 2011 NIH R01 grant $ 2,000,000 2006 – 2011 NSF Biophysics Grant $ 1,000,000 2006 – 2010 NIH R01 grant $ 800,000

Patents etc. (2008-2014) HIGH-DEFINITION DNA IN SITU HYBRIDIZATION (HD-FISH) COMPOSITIONS AND METHODS Filing Date: August 30, 2013 Based on U.S. Patent Application No(s).: 61/696096

- 107 - Previous research

Phenotypic variation is ubiquitous in biology and is often traceable to underlying genetic and environmental variation. However, even genetically identical organisms in homogenous environments vary, suggesting that random processes may play an important role in generating phenotypic diversity. The overarching goal of my laboratory is to the understand how stochastic gene expression is controlled, or utilized, during development and stem cell differentiation using the nematode worm Caenorhabditis elegans, embryonic stem cells, and the mammalian intestine as the main experimental model systems. My laboratory uses an integrated combination of quantitative experiments, theoretical and computational approaches, and the development of novel technology to develop a quantitative understanding of the origins and consequences of stochastic gene expression. My laboratory has a longstanding interest in stochastic gene expression since the start of my laboratory in 2000 at MIT.

Our first paper was a theoretical paper published in 2001 in PNAS. In this work we used theoretical tools from statistical physics to calculate the magnitude of the fluctuations that are expected based on the stochastic nature of transcription, translation and mRNA and protein degradation. In this work we presented testable predictions that inspired us to design experiments to validate the main model of the paper. The experimental follow-up was published one year later in Nature Genetics. Both the theoretical and experimental study have made a big impact in the field (these two papers together accumulated over 2000 citations, as reported by Google Scholar) and created a surge of interest in stochastic gene expression. After exploring the fluctuations in the expression of a single gene we started to explore how stochastic gene expression is modulated in gene regulatory networks. We developed both the theoretical and experimental tools to monitor gene expression fluctuations in a synthetic cascade of genes (Science 2005). As a next step towards a quantitative understanding of stochastic gene expression in endogenous gene regulatory networks we shifted our attention to feedback regulation in gene networks. We showed how these networks define two distinct stable states through positive feedback regulation and how stochastic fluctuations in gene expression allow random transitions between these two states even in a constant environment. These results were published in Nature in 2004 for the lactose operon in E. coli and in Nature in 2005 for the galactose uptake network of S. cerevisiae. We then showed that gene expression fluctuations have important physiological consequence in particular in fluctuating environments. We developed a theoretical model for this in 2004 (Genetics) and published the experimental validation in 2008 in Nature Genetics.

More recently, my laboratory started to explore the role of stochastic gene expression in multicellular organisms (Cell 2013; Nature 2010) and stem cells (Nature 2009). We also applied these single-cell systems biology tools to problems in cell signalling (Science 2013; Science 2008; Cell 2009; Nature 2009a), circadian rhythms (Nature 2007; Science 2010; Cell 2010), population biology problems (Nature 2009b), and microRNAs (Molecular Cell 2008; Molecular Cell 2010; Nature Genetics 2011; Nature Genetics 2013).

Because of our interest in single-cell biology my laboratory also started to develop new methodology to quantify biological activity in single cells. This effort started in 2008 when we developed smFISH (single molecule RNA FISH), a technology that allows the detection of single mRNA molecules in intact single cells (Nature Methods 2008; Nature Protocols 2013). Recently we adapted this approach to detect DNA loci in single cells with high spatial resolution (Nature Methods 2013a). Additionally the sensitivity of the smFISH technology was optimized to detect single SNPs in transcripts and therefore allowing allele-specific transcript detection (Nature Methods 2013b). Recently we also managed to utilize the optimized smFISH protocols to sort single cells based on transcript abundance using a FACS machine (Nature Methods 2004a). In September 2012 when I started my laboratory at the Hubrecht Institute, I initiated a new line of research: single-cell genomics. During the last two years we have been developing and applying new methods to quantify the transcriptome and genome from single cells. As a first step, we developed experimental and computational methods to separate the biological variability in single-cell transcriptome measurements from the significant technical variability (Nature Methods 2014b). Based on these insight we recently developed tomo-seq (Cell 2014). While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Tomo-seq is a method where we combined traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish

- 108 - embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

Since the start of my laboratory in January 2000 many Ph.D. (27) and M.S. (4) students graduated under my supervision. Most of the students started an academic career and several are now leading their own labs. I am currently guiding 6 graduate students from the Cancer, Stem Cells, and Developmental Biology (CSD) PhD program in Utrecht. My lab hosted 11 post-doctoral researchers, most of them now have faculty positions at renowned universities and institutes in the US and Europe (e.g. UC Berkeley, MIT, U Penn, UC San Diego, Weizmann Institute, Karolinska Institute, Biozentrum Basel, Leiden University). Currently I am supervising 9 post-doctoral researchers.

In summary, our combined theoretical and experimental effort has resulted in many peer-reviewed publications in the leading research journals. Our work accumulated over ten thousand citations in the last 5 years (as reported by Google Scholar) and let to the invitation of many reviews (for example: Cell 2008, Cell 2011, Cell 2014, Science 2012, Nature Reviews Genetics 2009, Nature Reviews Genetics 2014, Nature Methods 2011).

Future research

In the coming years we would like to further expand our single-cell genomics/transcriptomics focus while retaining our strength in imaging technology and computational modeling. In particular we would like to concentrate on the following topics:

1. Spatially resolved transcriptomics The formation of spatially distinct gene expression domains is a ubiquitous process during metazoan development, and is fundamental for the meticulous patterning required to drive embryogenesis. Identifying genetic pathways and regulators that are active in well-defined regions of the embryo is crucial to understand the processes of embryonic axis formation, tissue specification, and organ development. As a consequence, many studies in different model organisms and tissues, and at different developmental stages, have focused on identifying spatial patterns of gene expression on a large scale. Since such studies rely on microscopy-based approaches like mRNA in situ hybridization or immunohistochemistry – which unavoidably investigate only one or a few genes per sample – screening spatial expression patterns of the entire transcriptome has so far been out of reach. Conversely, RNA sequencing has emerged as a powerful tool to study gene expression on the genome-wide level, but is unable to yield spatially resolved information. Gene expression analysis after cell sorting can be used to determine in cell-specific transcriptomes, but the spatial resolution as well as the number of different cell types that can be screened are limited. In situ sequencing of RNA in intact tissues has the potential to provide direct information about the spatial organization of the transcriptome. However, this method is currently restricted by low detection efficiency and has not yet been demonstrated to detect gene expression patterns in intact tissues.

We recently developed a method for spatially resolved transcriptomics based on cryo-sectioning, which allows the generation of genome-wide spatial expression maps in 3D (Cell 2014). In our approach we cryo-section individual zebrafish embryos into 50-100 thin slices, extract RNA from the individual sections, and make use of in vitro transcription for linear amplification of cDNA in order to minimize amplification biases. By sectioning individual embryos in three different directions, we measure RNA profiles along the three body axes. Using mathematical image reconstruction inspired by optical tomography techniques such as computed tomography, and taking into account the shape of the embryo as determined by microscopy, we then reconstruct spatial expression patterns in 3D on a transcriptome-wide level. We provide a comprehensive genome-wide 3D expression atlas at three different stages of early zebrafish development – shield stage, 10 somites stage and 15 somites stage, and we show in a proof-of-principle experiment using mouse embryonic forelimbs that the protocol can be applied to other model organisms and to isolated tissues or organs. We demonstrate that our approach can identify novel spatial expression patterns in the zebrafish embryo, and validate selected candidates by traditional in situ hybridization. Our method and database represent a powerful resource to identify candidate genes expressed in any specific pattern without the need for anatomical annotation.

- 109 -

In the future we would like to further optimize this technology to quantify subcellular distributions of transcripts at a genome-wide scale. We are particularly interested in applying tomo-seq to early mouse embryos. Cells in the early mouse embryo have long been thought to be identical. Indeed, up to the 16- or 32-cell stage, embryonic cells are morphologically undistinguishable and exhibit a very high degree of developmental plasticity. However, there is now increasing evidence for spatial patterning as early as the 4- and 8-cell stage, which might possibly be linked to even earlier spatial inhomogeneity at the 1- and 2-cell stage. However, spatial patterning of early mouse embryos has so far not been studied on a genome-wide level. We would like to explore spatial patterning of early mouse embryos genome-wide and on the single-cell level. This will allow us to identify the first transcriptional patterning events associated with early cell fate decisions and ultimately formation of the three germ layers. Additionally we aim to expand tomo-seq to include detection of microRNAs and genomic DNA (for example for SNP and CNV detection).

2. Integrating and developing novel single-cell sequencing methods Recently, single-cell genome sequencing, single-cell methylome sequencing, and single-cell transcriptome sequencing, have emerged as promising tools to quantify genetic, epigenetic, and expression variability between individual cells, respectively. However, as these single-cell technologies are limited to quantifying the genome, the epigenome, or the transcriptome, it is currently impossible to explore the relation between these “omes” in single cells. We are working on integrating several single-cell “omics” methods to explore for example the relation between DNA copy number and transcript level in single cells or the relation between hydroxymethylation and transcription in single cells. Additionally we are very interested in combining these methods with sequence-based lineage tracing. Using Cre/lox or Cas9 technology we will induce DNA or mRNA barcodes that will genetically mark individual cells with different unique heritable codes. These codes can then be used to infer the lineage history for individual cells. We envision that we can induce over 1000 different barcodes.

3. Unbiased cell-type discovery using single-cell sequencing methods Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating sub-populations of cells rely on mRNA or protein expression of only few marker genes. The unequivocal identification of a specific marker gene, however, relies on the ability to purify a cell type, and thus poses a major challenge. The recent progress in single cell sequencing now permits an unbiased approach for identifying the entire spectrum of cell types within an organ based on their transcriptome. In collaboration with the Clevers lab, we recently started to sequence hundreds of randomly selected cells from mouse intestinal organoids. Since available computational methods can only resolve more abundant cell types, we developed RACEID, an algorithm for the rare cell type identification in mixed populations of sequenced single cells. We demonstrate that the algorithm can resolve cell types represented by only a single cell in our organoid sample. We plan to use and expand RACEID to identify novel marker genes of enteroendocrine cells, a rare population of secretory intestinal cells. Additionally we would like to explore how many cell types can be distinguished in the mouse intestine. Preliminary data suggest the presence of a surprising heterogeneous pool of enteroendocrine cells, expressing complex cocktails of hormones. We would like to apply these methods to other organs (e.g. human pancreas in collaboration with the de Koning lab; blood in collaboration with the Robin lab) in the near future.

Societal relevance and societal impact (2008-2014)

Understanding heterogeneity in gene expression and DNA copy numbers at the single-cell level is crucial to understanding treatments for diseases such as cancer. Tumors are intrinsically heterogeneous and it has been suggested that this variability is key to understanding the development of resistance to cancer therapy. We recently started to collaborate with clinical cancer biology labs (for example the Polyak lab and Chiarle lab at the Harvard Medical School) to apply our quantitative methods to tumors (Cell Reports 2014a, Cell Reports 2014b, Cancer Research 2014, Cancer Blood Journal 2014). From these studies we learned that tumor heterogeneity is a potent predictor for the success of the therapy. This work suggests the feasibility of predicting tumor evolution during therapy, which can be used to design more effective therapy strategies.

- 110 - The van Oudenaarden lab has been developing new methods for quantifying biological activity in single cells since 2008. Two of these novel methods have been commercialized by Biosearch Technologies, a company in California specialized in detection of nucleic acids [www.biosearchtech.com]. The first product (RNA FISH Stellaris) is based on the 2008 Nature Methods paper [Raj et al. Nature Methods 5, 877 (2008)], which allows the detection of single mRNA molecules in situ. This product turned recently into one of the most successful products of Biosearch Technologies. More recently Biosearch Technologies licensed the HD-FISH technology developed by the van Oudenaarden lab and described in their 2013 Nature Methods paper [Bienko et al., Nature Methods 10, 122 (2013)]. This novel DNA FISH technology allows detection of short DNA stretches with high spatial resolution. The van Oudenaarden lab is continuously in contact with Biosearch Technologies and other companies to explore commercialization of their new methods. This commercialization step is crucial to make these new methods available to a large community of scientists, clinicians, and companies.

In addition to leading a research lab, I have always been interested in education and mentoring. I am particularly interested in bridging the gap between the quantitative sciences such as mathematics and physics and the life sciences. I created a popular MIT Graduate course in Systems Biology (http://web.mit.edu/biophysics/sbio). This course was offered annually (2002 - 2009) and provided an introduction to the mathematical tools that are used to model the dynamics of gene and protein networks. In 2008, I received the prestigious School of Science Prize for Excellence in Graduating Teaching for creating and lecturing this course.

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- 112 -

Jacco van Rheenen

Key publications (2008-2014)

Ritsma L, Ellenbroek SIJ, Zomer A, Snippert HJ, de Sauvage FJ, Simons BD, Clevers H, van Rheenen J. (2014) Intestinal crypt homeostasis revealed at single stem cell level by in vivo live- imaging, Nature, 507(7492):362-3

Ellenbroek SIJ, van Rheenen J, (2014) Imaging hallmarks of cancer in living mice, invited submission to Nature Reviews Cancer, 14(6):406-18.

Ritsma L, Steller EJA, Beerling E, Loomans CJM, Zomer A, Gerlach C, Vrisekoop N, Seinstra D, van Gurp L, Schäfer R, Raats DA, de Graaff A, Schumacher TN, de Koning EJP, Borel Rinkes IH, Kranenburg O and van Rheenen J,(2012) Intravital Microscopy through an Abdominal Imaging Window Reveals Steps during Liver Metastasis, Science Translational Medicine. 4(158):158ra145

Jalink K and van Rheenen J.(2010) Nano-imaging of membrane topography affects interpretations in cell biology. Nature Methods, 7(7):486

Kedrin K, Gligorijevic B, Wyckoff J, Verkhusha VV, Condeelis J, Segall JE, van Rheenen J. (2008) Intravital imaging of metastatic behavior through a Mammary Imaging Window. Nature Methods, 5(12): 1019-21.

- 113 -

Prof. Dr. Jacco van Rheenen Cancer Biophysics

Group members Postdocs: Rinske Drost, Maria Alieva, Saskia Ellenbroek Graduate students: Arianna Fumagalli, Anoek Zomer, Danielle Seinstra, Sander Steenbeek, Colinda Scheele, Carrie Maynard, Lotte Bruens Technicians: Anko de Graaff, Ronny Schaefer, Evelyne Beerling

Curriculum vitae group leader

Name: Jacco van Rheenen Date of birth: 12 January 1978 Nationality: Dutch

Education/positions 2014-present Professor in Intravital Microscopy, University Medical Center Utrecht 2012-present Group leader (tenured), Hubrecht Institute 2008-2012 Junior group leader, Hubrecht Institute 2006-2008 Postdoc, Albert Einstein College of Medicine, Yeshiva University, Bronx, USA. For this research I obtained a fundamental and (pre) clinical cancer research from the Dutch Cancer Society (KWF). 2005-2006 Postdoc, Netherlands Cancer Institute 2000-2005 PhD in Biophysics, Netherlands Cancer Institute / Leiden University 1996-2000 MSc in Biology, University of Amsterdam

Memberships Dutch association of Cell biology American Association for Cancer Research

Awards 2014 Best seminar of 2014 of the research colloquium Cardiology at the UMCU 2013 Stem Cells Young Investigator Award 2013 (10,000 dollar personal money) 2012 Joined the editorial board of IntraVital, a journal that focuses on studies that visualize biological processes in multicellular organisms 2008 Awarded a VIDI grant by the Netherlands Organisation for Scientific Research (NWO). This is the most prestigious grant for starting group leaders in the Netherlands. 2006 Fellowship for fundamental and (pre-)clinical cancer research from the Dutch Cancer Society (KWF). Intravital imaging of metastasis and the immune responses at single cell resolution. 1999 Scholarship from the Bekker-la Bastidefonds for final rotation for Master Study at the University of Cambridge, UK 1999 Scholarship from the Faculteit Biologie fonds for final rotation for Master Study at the University of Cambridge, UK 1999 Scholarship from the Schuurman Schimmel-vanOuteren fonds for final rotation for Master Study at the University of Cambridge, UK

Other activities • In the editorial board of IntraVital (a journal that focuses on studies that visualize biological processes in multicellular organisms). • Chairman of the seminar series of the research school Cancer, Stem Cells and Developmental Biology • Organizer of the Master Class of the Cancer, Stem Cells and Developmental Biology, 2013, 2014 • Chair of the symposium “EVs in cancer metastasis” of the International Society for extracellular vesicles meeting, 2014 • Chair of the joint Dutch Society for Cell Biology/German Society for Cell Biology plenary on " Stem cells and cell fate" at the DGZ International meeting in Cologne in 2015.

- 114 - Thesis advisor for Laila Ritsma (2013)

Invited speaker on meetings (2008-2014) 2014 TropenInstituut meeting “Molecular Mechanisms in Cancer", Amsterdam 2014 Visualizing and Modeling Cellular and Sub-Cellular Phenomena, Ohio, USA 2014 Gray Institute for Radiation Oncology & Biology, University of Oxford, UK 2014 Massachusetts Institute of Technology, Boston, USA 2014 Applied Molecular Imaging Erasmus MC translational workshop, Rotterdam 2014 The ‘Genes & Cancer’ 30th Anniversary Meeting, Robinson College, Cambridge, UK 2014 Advanced imaging techniques: From bench to bedside, Rotterdam 2014 The Mammary Gland Biology Gordon Research Conference, Lucca, Italy 2014 The DFG Cancer Meeting of the „Hinterzartener Kreis für Krebsforschung“ at Villa Collina/Cadenabbia, Lake Como, Italy 2014 EMBO | EMBL Symposium Epithelia: the building blocks of multicellularity, Heidelberg, Germany 2014 Symposium Intravital Microscopy, Karolinska Institutet, Stockholm, Sweden 2014 DutchBiophysics, Veldhoven 2014 Dutch Physical Society, Leiden 2014 The course of European Calcified Tissue Society, Oxford, UK 2013 Imperial College London, UK 2013 University of Birmingham, Birmingham, UK 2013 University of Lausanne, Switzerland 2013 The Hematopoietic, Leukemic and Cancer Stem Cells meeting, Paris, France 2013 The Frontiers of Science of the Low Countries, Rotterdam 2013 Speaker at the workshop on 3D culture models for radiation and cancer research, Rotterdam 2013 Cancer Center Karolinska, Stockholm, Sweden 2013 The Friedrich Miescher Institute, Basel, Switzerland 2013 The 7th Winter Conference of the European Society for Molecular Imaging "Imaging Hallmarks of Cancer, Les Houches France 2012 Uppsala University, Uppsala, Sweden 2012 CBG/CGC KIT meeting, Amsterdam 2012 National Cancer Research Institute Cancer Conference, Liverpool, UK 2012 The 2nd Trippenhuis meeting “Dynamic Interactions in Cell Biology”, Amsterdam 2012 The FIRC Institute of Molecular Oncology Foundation-IEO Campus, Milan, Italy 2012 The 6th Imaging the cell, Toulouse, France 2012 The international liver congress, Barcelona, Spain 2012 The 4th course of Cytoskeleton, Paris, France 2012 The Pharmacology congress of the German society for Experimental and Clinical Pharmacology and Toxicology, Dresden, Germany 2012 The FLIM and FRET course Lamberts Instruments, Amstelveen 2012 Amsterdam Medical Center, Amsterdam 2012 Utrecht Microscopy Winter meeting, Utrecht 2011 Frontiers in imaging webcast, Leica 2011 The meeting Imaging Biocomplexity, Utrecht 2011 Institute for molecular and cell biology, Porto, Portugal 2011 The Zoo meeting, Amsterdam 2011 The American Association of Cancer Research, Orlando, USA 2011 Oxford University, Oxford, UK 2011 ELMI meeting, Alexandroupolis, Greece 2011 The LSR EU Kick Off Meeting in Mannheim, Germany 2011 Netherlands Cancer Institute, Amsterdam 2010 The NVvC meeting, Amsterdam 2010 Cancer Research UK, London, UK 2010 Breakthrough Breast Cancer, London, UK 2010 University of Nijmegen, Nijmegen 2010 Sonderforschungsbereich meeting, Munster, Germany 2010 University of Groningen, Groningen 2010 The meeting of European network of Breast development and cancer Labs, Weggis, Switserland

- 115 - 2010 Friedrich Miescher Institute, Basel, Switzerland 2009 Institute of integrated cell-material sciences, Kyoto University, Japan 2009 The Japanese Cell Biology meeting in Kobe, Japan 2009 The Institute of Biotechnology, University of Helsinki, Finland 2009 The Institute for Molecular and Cellular anatomy, Universitatsklinikum Aachen, Germany 2008 The meeting for the Dutch Society of Cell Biology, Utrecht 2008 Cell Migration Consortium, Imaging and Photomanipulation, Washington DC, USA

Grants (2008-2014) 2014 NWO Earth and Life Sciences Open Program: “Identifying the physiological relevance of RNA transfer by microvesicles”. € 240,000 2013 NWO Gravitation, participant of the Cancer Genomics Centre Netherlands (CGC.nl). € 450,000 2012 Research grant from the Association for International Cancer Research (AICR): “Intravital lineage tracing of (cancer) stem cells in genetic mouse models”. € 240,000 2010 A NWO middel-groot equipment grant from the Netherlands Organisation for Scientific Research: A spinning disk confocal microscope to image epithelial and endothelial development, tumor formation and metastasis. € 300,000 as co-PI 2009 VIDI personal grant from the Netherlands Organisation for Scientific Research: Influence of extracellular matrix remodeling by stromal cells on invasion and intravasation of mammary tumor cells. € 800,000 2009 Research grant for fundamental and (pre)-clinical cancer research from the Dutch CancerSociety (KWF): MenaINV-induced EGFR clustering causes mammary carcinoma cells to become invasive. € 600,000 2008 A NWO groot equipment grant from the Netherlands Organisation for Scientific Research: A two-photon microscope containing two infrared lasers to excite cyan, green, yellow and red fluorophores in living animals. € 1,000,000

Patents etc. (2008-2014) 2010 In vivo quantitative screening test for anti-metastasis treatment efficacy (US patent appl 20110296538)

Previous research

Most of our knowledge of tissue homeostasis, tumor initiation and tumor progression is derived from techniques that draw a static picture of these highly dynamic processes. To study the dynamic aspects of these processes, in 2008 I established a research group that develops state-of-the-art imaging techniques to visualize and study individual cells in real-time in living animals, referred to as intravital microscopy. We have crossed the latest lineage tracing mouse models and cell type-specific fluorescent mouse models with genetic tumor mouse models and combined these with intravital microscopy to study the migratory properties and fate of cells, with a focus on stem cells, during tissue homeostasis and tumor initiation and progression.

Gaining prolonged optical access to healthy and tumorigenic tissues through our imaging windows: In the intravital imaging field it is common to surgically expose tissues that are located deep inside animals such as breast, intestinal and liver tissue, which limits intravital microscopy experiments to a few hours. However, we aspired to study the dynamic processes of tissue homeostasis and tumor progression, which requires both intravital microscopy and visual access to tissue over prolonged times. Therefore, we invented the mammary imaging window (Kedrin et al., Nat Meth, 2008) to intravitally visualize breast tumors, and the abdominal imaging window (Ritsma et al., Sci Trans Med, 2012) to intravitally visualize intestines and liver metastases. To study the behavior and fate of individual cells over multiple days to weeks, we also developed methods to label and trace individual cells and retrace imaging fields over multiple imaging sessions. These developments have been big breakthroughs in the intravital microscopy field and are now widely adopted by other labs (e.g. each month we train researchers to get acquainted with our technology). Our unique approach led to many discoveries that could not be extracted by any other technique (a few example studies are highlighted below).

- 116 - Intestinal crypt homeostasis revealed at single stem cell level by in vivo live imaging: Standard lineage tracing experiments showed that Lgr5+ crypt base columnar (CBC) cells generate all intestinal epithelial lineages, and identified Lgr5+ cells as stem cells of the small intestine and colon. However, these lineage tracing experiments lack the crucial information about the history of the lineage at the time of measurement. For example, these lineage tracing experiments cannot resolve whether individual Lgr5+ cells positioned at different locations within the crypt base compartment display different behavior, since the epithelial lineage cannot be related to the individual Lgr5+ stem cell. To overcome this, we have developed a novel approach for continuous intravital imaging of Lgr5- Confetti mice (Ritsma et al., Nature 2014). Using this approach we have shown that Lgr5+ cells in the upper part of the stem cell niche (termed 'border cells') can be passively displaced into the transit- amplifying (TA) domain, following division of proximate cells, implying that determination of stem cell fate can be uncoupled from division. Through the quantitative analysis of individual clonal lineages over time, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings established a novel paradigm for stem cell maintenance in which a dynamically heterogeneous cell population is able to function long-term as a single stem cell pool (Ritsma et al., Nature 2014).

Intravital imaging of cancer stem cell plasticity in mammary tumors: It is widely debated whether all tumor cells within a tumor have the same potential to propagate and maintain tumor growth, or whether there is a hierarchical organization. Lineage tracing experiments have recently shown the existence of small populations of cells, referred to as cancer stem cells (CSCs), that maintain and provide growth of squamous skin tumors and intestinal adenomas. Using intravital lineage tracing experiments, we have shown that mammary adenomas and carcinomas (in the genetic PyMT model) also contain CSCs (Zomer et al., Stem Cells 2013). However, in contrast to the aforementioned lineage tracing techniques which provide static images and lack the ability to study whether stem cell properties can be obtained or lost, the use of intravital lineage tracing in our study demonstrated the existence of this process referred to as stem cell plasticity. Our data indicate that existing CSCs disappear and new CSCs form during mammary tumor growth, illustrating the dynamic nature of these cells (Zomer et al., Stem Cells 2013).

Intravital imaging of dissemination and metastasis formation Complications caused by metastases are the major cause of cancer-related death. Metastasis is a multistep process in which only a minority of cells within a tumor acquire traits and are surrounded by microenvironments that enable them to disseminate and form distant metastases. Using intravital microscopy, we dissected various critical steps of metastasis, and monitored the dynamic processes that are involved in the rare individual cells that metastasize. For example, we have shown that tumor cells surrounding major blood vessels are motile, intravasate and get transported to the lungs, in contrast to tumor cells surrounding microvessels, which only display intratumoral motility (Kedrin et al., Nat Meth 2008). Moreover, we showed that the presence of T cells in the microenvironment supports the motile behavior of mammary tumors (Ritsma et al., Nat Comm 2013). Furthermore, our abdominal imaging window enabled us to visualize and study the formation of metastases from individual cells that arrived in the liver (Ritsma et al, Sci Trans Med 2012). We showed that single extravasated tumor cells proliferate to form “pre-micrometastases”, in which cells are motile. Genetically and chemically suppression of this motility reduces the metastatic load by 50%, suggesting that tumor cell migration is an important for the formation of liver metastases.

Future research

Using our intravital imaging approach and our different mouse models, we will continue studying how cells gain or lose stemness and migratory properties during tissue homeostasis, tumor initiation, and tumor progression. We have created several new fluorescent mouse models in which cells with migratory and/or stem cell properties are specifically fluorescently marked. In addition to the ability to study these populations of cells by intravital microscopy, the fluorescent labeling enables us to isolate, molecular characterize, manipulate, and transplant these small but dangerous populations of cells. Our current studies are focused on three subjects: 1) Determine the role of stem cell plasticity in tissue homeostasis, tumor initiation and tumor progression. 2) Establish the role of microvesicle transfer in tumor heterogeneity and tumor progression.

- 117 - 3) Reveal the role of EMT and MET in migration, metastasis and chemotherapy resistance.

Determine the role of stem cell plasticity in tissue homeostasis, tumor initiation and tumor progression By intravital imaging, we elucidate whether the location of stem cells within the crypt base influences their competitional strength when acquiring tumor-initiating mutations. For example, we are currently studying whether the competitional advantage that stem cells get upon loss of one of the APC alleles may be lost or intensified when the cell’s position within the niche is altered. In addition to intestinal tissues, we have started similar studies in breast tissue.

We also continue to work on the role of cancer stem cells in tumor growth and metastasis in mammary tumor models. We have created mouse models and designed experiments to identify and isolate cancer stem cells. Based on single cell mRNA sequencing, we will molecularly characterize these cells. If we successfully identify a marker, we will generate mice with an inducible GFP-CreERT2 knock-in allele, and perform intravital lineage tracing experiments to directly study plasticity in cancer stem cells and subsequent lineages. Moreover, we use our current intravital lineage tracing approach to measure the kinetics of stem cell plasticity, tumor proliferation and cell death in various tumor models. Based on these data, we are building a Monte Carlo computer model that describes the growth of our tumors at cellular level, which will ultimately help to predict successful targeting of tumor growth.

With our invention of the abdominal imaging window we study primary colorectal tumors and liver metastases. We have successfully generated a fluorescent mouse model that forms colorectal carcinomas that spontaneously metastasize to the liver (based on the loss of APC and P53 and expression of KRAS). In addition to studying the role of cancer stem cells in tumor growth, we study whether cells need to acquire stemness properties in order to migrate and escape from the primary tumor and colonize the liver. For example, we study whether and how Lgr5+ and Lgr5- tumor cells are able to grow liver metastases.

Establish the role of microvesicle transfer in tumor heterogeneity and tumor progression. Using the Cre-Lox system and intravital imaging, we have shown that malignant tumor cells can transfer mRNA and proteins through microvesicles to more benign cells locally and systemically thereby enhancing the migratory and metastatic capacity of the recipient cells (Zomer et al, in progress for resubmission at Cell). We are currently generating mouse models to study the potential transfer of mRNA and proteins between healthy and tumor cells. Moreover, we are performing experiments to molecularly characterize cells that release and/or take up microvesicles, in order to design strategies to target microvesicle exchange. All this work will increase our fundamental knowledge, for example on how metastasized cells influence the metastatic capacity of more benign tumor cells in the primary tumor.

Reveal the role of EMT and MET in migration, metastasis and chemotherapy resistance To study the role of epithelial to mesenchymal transition (EMT) in tumor cell migration and metastasis, we have generated a genetic fluorescent PyMT mouse model in which tumor cells that undergo EMT (or the reverse process MET) change color. Using intravital microscopy, we visualize and study these processes at several stages of metastasis in real time. Moreover, we isolate cells with various EMT states during different stages of metastasis by flow cytometry, and characterize these cells by single cell sequencing, genetic manipulation, and transplantation studies. By these studies, we will shine light on the involvement of EMT and MET in the various steps of metastasis, and identify new markers for migratory and metastatic cells.

We also use this mouse model to test whether EMT is important for development of chemotherapy resistance. Chemotherapy, such as cisplatin, induces tumor cell death, but some tumor cells survive treatment and cause regrowth of the tumor. By intravital microscopy and flow cytometry, we have shown that migratory EMT cells get enriched in tumors that regress upon therapy, which is in line with the idea that EMT cells contain stem cell properties that enable them to better resist chemotherapy. By a variety of experiments including intravital microscopy, single cell sequencing and transplantation studies, we are currently testing whether migratory EMT cells get enriched because these cells are either more resistant to therapy, or that EMT/migration is induced upon treatment. With this line of research, we will reveal the role of EMT and MET in migration, metastasis and chemotherapy resistance.

- 118 - Societal relevance and societal impact (2008-2014)

Cancer affects the lives of thousands of patients Statistically, 30% of the population gets affected by cancer directly as a patient or indirectly through their family. In the Netherlands, yearly more than 100.000 patients are diagnosed with cancer. Patients that are diagnosed early with a benign tumor can be surgically treated, and the impact is relatively minor. However, many types of cancer, especially when detected at later stages when primary tumors metastasize, are difficult to treat.

Knowledge of stem cell plasticity may be important for the design of future therapies The most common treatment strategy for cancer is surgical removal combined with chemotherapy. Chemotherapy is often given prior to surgery to reduce tumor size in order to improve the success rate of tumor resection and after surgery to reduce potential metastasis. Despite decades of research and introduction of new therapeutics, cancer treatment is still inefficient: only one out of four patients benefits from anti-cancer drugs. Therapeutics targeting the “average” differentiated cell may potentially fail to target the “non-average” population of invasive and stem cells that are responsible for the formation and spread of tumors. So far, it has been difficult to study this “non-average” population of tumor cells, since commonly-used technology, such as histochemistry, (q)PCR and Western blotting, provide snapshots of large populations of cells and therefore fail to provide crucial information about the history of individual cells (e.g. the few surviving cells amongst the majority of dying cells in a regressing tumor). During the last few years, we have developed imaging techniques and fluorescent mouse models that enabled us to specifically study these “non-average” populations of cells and gained fundamental knowledge on their behavior during homeostasis, growth and metastasis. We showed that “new” stem cells may be formed by dedifferentiation of differentiated tumor cells that get located into a stem cell-inducing microenvironment (stem cell niche). Moreover, we showed that tumor cell migration is highly dependent on the surrounding microenvironment. Chemotherapy can potentially change microenvironments –and therefore potentially stem cell/migratory niches- with non- predictable consequences for the number of migratory and cancer stem cells and subsequently for the outcome of cancer treatment. Although the fundamental knowledge we obtained may not immediately and directly impact on patients, this type of knowledge is key for the design of new types of strategies to target cancer.

Future extension to human samples Our research mainly focuses on pre-clinical mouse models that recapitulate human tumors. We have set up collaborations to extend our research towards human samples. For example, we collaborate with pathologist Prof. Dr. Paul van Diest from the University Medical Center Utrecht, the Netherlands. Through this collaboration, we obtain breast tumor tissue from patients. Similar to our experiments in pre-clinical mouse models, in these human samples we are currently trying to isolate EMT-cells from lobes of tumors that stain positive for E-cadherin, and try to identify the expression profile of these “non-average” cells. In collaboration with Prof. Dr. Emile Voest, medical director of the Netherlands Cancer Institute, we are setting up similar studies on samples of patients before and after chemotherapy treatment. These collaborations help us to clinically translate our findings in mice.

In addition to these collaborations, the van Rheenen lab is member of the Cancer GenomiCs.nl (CGC.nl), which is a consortium of prominent cancer research groups from seven research institutions in the Netherlands. The mission of the CGC.nl consortium is to determine and understand genetic alterations in individual tumors in order to deliver precision medicine to individual cancer patients. Our ambition is to significantly improve life expectancy and quality of life for cancer patients and to provide multidisciplinary training for the next generation of cancer researchers and specialists. The scientists were selected based on their excellence and their commitment to the mission of the program. Within this consortium, the van Rheenen lab collaborates with the groups of Prof. Dr. Rene Bernards, Prof. Dr. Rene Medema and Prof. Dr. Hans Bos. In this collaboration, we use our intravital imaging expertise to establish drug responses of tumors at a cellular resolution. These collaborations will also further help us to clinically translate our findings and techniques.

- 119 - Communicating science to society I strongly believe that scientists should, in addition to excel in their own discipline, engage the general public in their work. Since the society should decide on the ethical boundaries of science, it is essential to explain the aims and outcomes of science in layman’s language. In addition, to secure the quality of science in the Netherlands, it is of utmost importance to stimulate the young generation to be interested in science and becoming a scientist. Lastly, although biomedical science is highly important for future medicine, it is also extremely costly. To convince society to keep investing in science, the general public should be informed on the progress we make. In the last couple of years, I have been involved in explaining about cancer research and engaged in the public debate about science in general. My work attracted a lot of media attention, including all national newspapers (nu.nl, Volkskrant, Telegraaf etc), a lot of international newspapers (Newscientist, The Scientist, Elmundo, Dagensmedicin, etc), radio programs (BBC, Radio 5, etc), and television programs. (RTL4 etc). This attention gave me the opportunity to explain in layman’s language why my work is so important and how we have contributed to the understanding of cancer. The positive feedback I received (including re-invitations) let me strongly believe that I can explain the fun and importance of science to the general public. In addition, I have enjoyed helping “De Jonge Akademie on Wheels” and have given lectures at highschools (e.g. de Breul) to stimulate kids to think about science and a scientific career.

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Eva van Rooij

Key publications (2008-2014) van Rooij E, Quiat D, Johnson BA, Sutherland LB, Qi X, Richardson JA, Kelm Jr RJ, and Olson EN. (2009) A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance. Dev Cell. 17(5):662-73

Montgomery RL, Hullinger TG, Semus HM, Dickinson BA, Seto AG, Lynch JM, Stack C, Latimer PA, Olson EN, van Rooij E. (2011) Therapeutic inhibition of miR-208 improves cardiac function and remodeling during heart failure. Circulation. 4;124(14):1537-47.

Hullinger TG, Montgomery RL, Seto AG, Dickinson BA, Lynch JM, Stack C, Latimer P, Dalby CM, Robinson KH, Hare J, Olson EN and van Rooij E.(2012) LNA-mediated inhibition of miR-15 protects against cardiac ischemic injury. Circ Res. 110(1):71-81.

Grueter CE, van Rooij E, Johnson BA, Deleon SM, Sutherland LB, Qi X, Gautron L, Elmquist JK, Bassel-Duby R, Olson EN. (2012) A Cardiac MicroRNA Governs Systemic Energy Homeostasis by Regulation of MED13. Cell 149(3):671-83.

Montgomery RL, Yu G, Latimer PA, Stack C, Robinson K, Dalby CM, Kaminski N, van Rooij E. MicroRNA mimicry blocks pulmonary fibrosis. EMBO Mol Med. 2014 Sep 19 Epub.

- 121 - Dr. Eva van Rooij Molecular Cardiology

Group members Postdocs: Monika Gladka-de Vries, Farhad Akbari Moqadam, Gregory Lacraz, Anne Katrine Johansen Graduate students: Charlotte Demkes, Bas Molenaar, Koen Scholman, Joep Eding Technicians: Hesther de Ruiter, Danielle Versteeg

Curriculum vitae group leader

Name: Eva van Rooij Date of birth: 1 July 1977 Nationality: Dutch

Education/positions 1996 - 2000 MSc, University Hospital Maastricht 2001 - 2004 PhD, University Hospital Maastricht – Hubrecht Institute 2005 - 2007 Postdoctoral fellow in the lab of Prof E.N. Olson, Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas. USA 2007 - 2009 Instructor in the lab of Prof. E.N. Olson, Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas. USA 2007 - 2009 Adjunct Director of Research and scientific co-founder of miRagen Therapeutics, Boulder, CO, USA 2009 - 2011 Director of Biology at miRagen Therapeutics, Boulder, CO, USA 2011 - 2013 Senior Director of Biology at miRagen Therapeutics, Boulder, CO, USA 2013 – present Special Advisor and scientific co-founder miRagen Therapeutics, Boulder, CO, USA 2013 – present Junior group leader and Associate Professor Hubrecht Institute KNAW and University Medical Center Utrecht

Memberships - European Council of Cardiology (Heart failure council) - American Heart Association - International Society of Heart Research (European council)

Awards 2007 American Association for the Advancement of Science/Science Program for Excellence in Science, UT Southwestern Medical Center Dallas, Texas. 2007 ISHR Young Investigator Award, June 2007, Bologna Italy 2008 Finalist Young Investigator Louis N. & Arnold Katz award, Nov 11 2008, at Basic Science American Heart Association meeting New Orleans, USA 2011 AHA council for Basic cardiovascular Sciences award for most downloaded review article from Circulation research. 2012 Circulation Research, Best manuscript award. 2014 Outstanding Achievement Award from the European Society of Cardiology (ESC) Council on Basic Cardiovascular Science

Other activities • Member of the Cancer, Stem cells & Development seminar committee UMCU

2009- current Editorial Board Circulation Research 2009- 2013 Associate member transatlantic Leducq network: miRNAs in cardiovascular disease 2011 Organizer Keystone Meeting, “MicroRNAs and Human Disease” 2011 Guest Editor for Circulation Research. 2013- current Scientific Advisory Board member miRagen Therapeutics, Inc. 2013- current Heart Failure Association Committee European Society (ESC) of Cardiology 2013- current Editorial board Journal of Molecular and Cellular Cardiology 2014- current Editorial board Molecular Therapy

- 122 - 2014 Co-Organizer, Cardiology workshop ESCI 2014 Member of ESC working group on Myocardial Function 2014 International Society Heart Research European Council member

Thesis advisor for Due to my previous appointment in industry I so far have not mentored any PhD students, but am currently supervising 4 students that are expected to graduate in 2017-2018.

Invited speaker on meetings (2008-2014) 2008 Keystone Symposia Pathological and Physiological Regulation of Cardiac Hypertrophy 2008 microRNA in Human Disease and Development Cambridge Healthtech Institute, Cambridge 2008 Experimental Biology, San Diego 2008 American Heart Association Scientific Sessions, New Orleans (2 invited talks and finalist Katz award) 2009 Second Annual microRNAs in Human Disease meeting. Co-sponsored by Santaris Pharma, RxGen, and miRagen therapeutics, St Kitts 2009 Fourth Microsymposium on Small RNAs, Institute of Molecular Biotechnology, IMBA, Vienna 2009 microRNA in Human Disease and Development Cambridge Healthtech Institute, Cambridge 2009 Basic Cardiovascular Sciences Annual Conference 2009 - Molecular Mechanisms of Cardiovascular Disease 2009 Heart Failure Society of America (HFSA) 13th Annual Scientific Meeting. Boston 2009 American Heart Association Scientific Sessions, New Orleans, USA (3 invited talks) 2010 Third Annual microRNAs in Human Disease meeting. Co-sponsored by Santaris Pharma, RxGen, and miRagen Therapeutics, St Kitts 2010 RNAi Interference in Cardiovascular Disease for the Experimental Biology meeting, Anaheim (2 invited talks) 2010 Keynote lecture College of Pharmacy Research Day, Ohio State University 2010 Abcam meeting Birth, Life and Death of the Cardiac Myocyte, Napa Valley 2010 Heart Failure Congress 2007, Oslo, Norway 2010 American Heart Association Scientific Sessions, Chicago, USA (2 invited talks) 2010 Guest lecturer at Interdisciplinary Stem Cell Institute, University of Miami Health System, Miami 2011 Cardiovascular Drug Discovery, British heart foundation centre of research excellence symposium, London, UK 2011 Keystone symposia MicroRNAs and Human Disease (J6), Banff, Alberta (speaker & organizer) 2011 Keystone symposia Molecular cardiology: Disease Mechanisms and experimental Therapeutics, Keystone, Colorado 2011 Basic Cardiovascular Sciences Annual Conference 2011 - From Concept to Clinic – Leading Translational Cardiovascular Science, New Orleans 2011 Non-coding RNAs and human disease symposium, Copenhagen, Denmark. 2011 European Society of Cardiology meeting, Paris, France 2011 Heart Failure Society of America (HFSA) 15th Annual Scientific Meeting. Boston 2011 ASN Advances in Research Conference on microRNAs, Philadelphia 2011 American Heart Association Scientific Sessions, Orlando, USA (2 invited talks) 2011 Guest lecturer at San Diego State University 2012 microRNA in Human Disease and Development Cambridge Healthtech Institute, Cambridge 2012 Keystone symposia Fibrosis: Translation of Basic Research to Human Disease and Novel Therapeutics. Big Sky, Montana. 2012 DIA/FDA Oligonucleotide-based Therapeutics 2012: April 16-18, 2012 at the Washington Marriott Metro Center, Washington, DC. 2012 Experimental Biology, San Diego 2012 Cardiac Regulatory Mechanisms, Gordon Research Conference 2012 European Society of Cardiology meeting, Munich, Germany 2012 Advances in molecular mechanisms of Heart Failure and myocardial Repair & Natriuretic Peptide Research, August 2012, Oulu, Finland 2012 Guest lecturer at Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre 2012 American Heart Association Scientific Sessions, Los Angeles, USA (3 invited talks) 2013 Keystone symposia: Noncoding RNAs in Development and Cancer Vancouver, Canada.

- 123 - 2013 Heart Failure winter meeting, Diablerets, Switserland 2013 11th Dutch-German joint meeting of molecular Cardiology, Heidelberg, Germany 2013 International Dialogues in Cardiovascular Medicine, Cascais, Portugal 2013 Utrecht Stem Cell Conference, Utrecht, Netherlands 2013 Marabou symposium, Role of miRNA in Nutrition and Disease, Stockholm 2013 Basic Cardiovascular Sciences Annual Conference 2013 - From Concept to Clinic – Leading Translational Cardiovascular Science, Las Vegas 2013 Heart Failure Society of America (HFSA) 17th Annual Scientific Meeting. Orlando 2013 University lecture BHF Kings College 2014 Keystone symposia Molecular cardiology: Disease Mechanisms and experimental Therapeutics, Santa Fe 2014 Heart Failure winter meeting ESC, Diablerets, Switserland 2014 12th Dutch-German joint meeting of molecular Cardiology, Groningen, the Netherlands 2014 International Society of Heart Research, Miami 2014 European Society of Cardiology meeting, Barcelona, Spain 2014 Guest lecturer at Dutch Cancer Institute (NKI) 2014 American Heart Association Scientific Sessions, Chicago, USA

Grants (2008-2014) 2009 National Institutes of Health Phase I SBIR - 1R43HL099065-01 Title: Optimized chemistries for inhibition of pathological microRNAs in cardiac disease Total amount awarded: $ 807,240

2013 - 2017 Stimulerings Associate Professorship funding UMCU Total amount awarded: € 275,000 for 4 years

2013 - current Sponsored Research agreement through miRagen Inc. / Servier Total Award Amount: $ 200,000 per year

2014 - 2019 ERC consolidator grant, MicroRNA function in Cardiac and Metabolic disease Total amount awarded: € 2,000,000 for 5 years

2014 - 2018 Dalijn Foundation, MicroRNAs regulating cardiac contractility Total amount awarded: € 250,000 for 4 years

2014 - 2017 ARVD Foundation, MicroRNAs in ARVD Total amount awarded: € 506,000 for 3 years

2014 - 2019 Fondation Leducq, Enhancing cardiac regeneration Total amount awarded: € 608,000 for 5 years

Patents (2008-2014) 1. Olson, E.N., van Rooij, E. Identification of an miRNA that activates expression of beta-myosin heavy chain (miR-208). 2. Olson, E.N., van Rooij, E. MicroRNAs of the miR-15 family modulate cardiomyocyte survival and cardiac remodeling (miR-15a, -15b,-16, -195, -424 and -497). 3. Olson, E.N., van Rooij, E. MEF-2 dependent muscle specific miRNA that controls skeletal muscle fiber identity (miR-499). 4. Olson, E.N., van Rooij, E. MicroRNA family that modulates fibrosis and uses thereof (miR-29). 5. Olson, E.N., van Rooij, E. Identification of microRNAs that control myosin expression and myofiber identity (miR-208, -208b and -499). 6. Olson, E.N., van Rooij, E. Identification of miRNAs involved in post myocardial infarction remodeling. 7. Olson, E.N., van Rooij, E. Dual targeting of miR-208 and miR-499 in the treatment of cardiovascular disorders (miR-208, -208b and -499). 8. Olson, E.N., Quiat D., van Rooij, E. MicroRNA regulation in Ischemia/Reperfusion injury. 9. van Rooij, E., Dalby, C., Montgomery, R.L. Oligonucleotide-based inhibitors comprising locked nucleic acid motif

- 124 - 10. van Rooij, E., Dickinson, B.A., Seto, A.G. Blood-borne miRNAs as surrogate markers of drug efficacy for cardiac conditions 11. Olson, E.N., Grueter C., van Rooij, E. Suppression of obesity and normalization of insulin sensitivity by a microRNA inhibitor 12. van Rooij, E., Dalby, C., Seto, A.G. Inhibitors of the miR-15 family of microRNAs 13. Seto, A.G., van Rooij, E., Robinson, K.H., Dalby, C. Locked nucleic acid inhibitor of miR-145 and uses thereof.

Previous research

Despite a detailed understanding of the molecular and cellular processes governing cardiac function and contractility, cardiovascular disease remains a primary cause of morbidity and mortality worldwide. Although numerous treatment options like statins, ACE inhibitors, beta-blockers and others show therapeutic benefit, cardiovascular disease continues to increase in prevalence, underscoring the need for new therapeutic strategies. MicroRNAs are short, single-stranded non-coding RNAs that anneal with complementary sequences in mRNAs, thereby suppressing protein expression. Since individual microRNAs engage numerous mRNA targets that are often encoding multiple components of complex intracellular networks, the manipulation of a microRNA can have a profound impact on cellular phenotypes. In the last decade it has become increasingly clear that microRNAs are relevant players during disease, including cardiovascular disorders. In the last several years our research has focused on microRNA and gene function in heart disease. In doing so we were able to make seminal contributions to the field of microRNA biology for cardiovascular disorders. In 2006, we published a groundbreaking study that for the first time linked changes in microRNA expression to cardiac hypertrophy and heart failure. MicroRNA profiling studies in both animal models and human tissue samples of heart failure patients indicated that microRNAs are dynamically regulated during disease and that the regulation of the microRNAs actively influences the disease process (van Rooij et al. PNAS 2006). In a follow up paper we showed the relevance of additional microRNAs, like the miR-15 family, in the setting of ischemic heart disease (van Rooij et al. PNAS 2008). These findings pointed to previously unrecognized roles of microRNAs in the control of cardiac growth and function and opened up an entirely new avenue of investigation for the field. During our efforts to understand the influence of microRNAs on heart disease, we discovered that the gene encoding for one of the main contractile proteins of the heart, α−myosin heavy chain (MHC), intronically contains a microRNA, miR-208. Like αMHC, miR-208 is expressed exclusively in the heart, making it the sole cardiac-specific microRNA that has been annotated so far. Although genetic deletion of miR-208 in mice failed to induce an overt phenotype under baseline conditions, miR-208 knockout mice show virtually no signs of pathological cardiac remodeling (like hypertrophy of cardiomyocytes or fibrosis) and they are unable to upregulate βMHC expression (van Rooij et al. Science 2007). This is particularly important because even a subtle shift in the balance of αMHC versus βMHC expression toward βMHC reduces mechanical performance and efficiency of the adult heart. The known sequence of a microRNA and their heightened functions under conditions of pathophysiological stress and disease, make them attractive candidates for therapeutic manipulation. Lessons learned from antisense technologies catalyzed opportunities to therapeutically regulate microRNAs by using oligonucleotide chemistries, known as antimiRs. These modified antisense oligonucleotides can reduce the levels of pathogenic or aberrantly expressed microRNAs and have shown to be efficacious in both animals and humans. Because microRNAs typically act as inhibitors of gene expression, antimiRs will result in a de-repression of the mRNAs that are normally targeted by the microRNA. The rapidly growing knowledge on the functional relevance of microRNAs during heart disease, the shortage of effective therapies, and the ability to potently and specifically regulate microRNAs in vivo prompted me and others in 2007 to start a biotech company, called miRagen Therapeutics (http://www.miragentherapeutics.com). The goal of miRagen is to develop microRNA- based therapeutics for heart disease. After we founded miRagen Therapeutics Inc., I followed ‘my microRNAs’ and joined the company to serve as miRagen’s head of research. While managing our research team, I was responsible for guiding the scientific direction of the company, overseeing patent filings, and reporting to our board of directors. During this time I also took the lead in initiating and managing several academic and commercial collaborations. After defining the most potent chemistry to inhibit cardiac microRNAs, we started to explore the therapeutic value of some of our microRNA targets by trying to recapitulate the cardioprotective phenotypes we observed in our genetic models by using antimiRs.

- 125 - Based on the beneficial effects of miR-208 loss of function in knockout mice, we explored the effects of therapeutic regulation of miR-208 during cardiac disease. We showed that inhibition of miR-208 by systemic injection of antimiR-208 in a rat model of hypertension-induced heart failure suppressed hypertrophy, fibrosis, diminished βMHC expression and improved survival in a rat model of diastolic heart failure (Montgomery et al. Circ. 2013). Our profiling study in ischemic heart disease showed a strong induction of members of the miR-15 family in the heart in response to myocardial infarction, which causes death of cardiomyocytes and loss of pump function. Using an antimiR-based strategy were able to show that inhibition of the miR-15 family reduced infarct size and improved cardiac function in a model of ischemia reperfusion (Hullinger et al. Circ Res 2012), representing an intriguing strategy to enhance cardiac repair following injury.

However, with miRagen growing up as a result of our success, we started to move away from doing the kind of basic discovery science that I like doing so much. For this reason I returned in to academia in the beginning of 2013 and started building an academic research team at the Hubrecht Institute, while maintaining my connection with our company.

Our initial studies have focused on defining the cardiac function of a key downstream target of miR- 208, known as Zinc finger E-box binding homeobox 2 (ZEB2). Using mouse genetics (collaboration with Prof Huylebroeck, Erasmus MC) we are currently investigating the cardiac function of this gene under basal and stress conditions using both gain- and loss-of-funtion studies. Additionally we are exploring a microRNA cluster that might be relevant for cardiac contractility by the regulation of AC6. During heart failure there is a decline in cardiac contractility that is generally associated with a reduced expression or function of adenylyl cyclase (AC)6. AC is the enzyme that converts ATP to cAMP, which is an important regulator of cardiac muscle contraction Pharmacological pre-clinical animal studies have shown that increased levels of AC6, improves defective Ca2+ handling in the failing heart, and increases function of the aged heart. In search for microRNAs that are relevant for cardiac disease, we identified 4 conserved binding sites for miR-182 and miR-96 in the 3’-UTR sequence of AC6. Since the presence of multiple conserved binding sites for a single microRNA in the 3’UTR sequence of a gene implies a strong link between the microRNA and the expression of the target gene, we are currently exploring the relationship between these microRNAs and cardiac contractility in more detail.

Future research

Ischemic heart disease (IHD) plagues industrialized nations and can be held responsible for a large proportion of the health-care budget and resources. During an acute myocardial infarction (AMI) roughly 1 billion cardiomyocytes, which reflect on average 20% of the total cardiomyocyte fraction of the heart, are lost after an averaged sized MI. Since cardiomyocytes are terminally differentiated cells, the lost tissue is not restored. An AMI therefore often causes a chronic condition that can induce adverse cardiac remodeling and eventually lead to congestive heart failure. Given the global burden of ischemic heart disease and its increasing prevalence in aging populations, the development of strategies to regenerate the human heart is among the most important challenges facing human health.

In the past decade, there has been great excitement around the concept of cardiac regeneration through delivery of exogenous stem cells, although the results so far have not lived up to the expectations. The development of a new, effective treatment options that can minimize the loss of cardiomyocytes and/or reverse the adverse remodeling processes in the post-MI heart would be of great value for a large number of patients.

While the heart is notoriously resistant to regeneration, considerable evidence suggests that the fundamental biology of the myocardium provides multiple therapeutic opportunities to enhance cardiac regeneration. A major future focus of our lab will to explore opportunities to stimulate or boost the repair mechanisms that are endogenously present in the heart, with the ultimate aim to enhance cardiac regeneration after an ischemic insult to maintain a better cardiac output. In doing so we will focus on 3 main aspects that could aid cardiac repair: - Enhancing cardiomyocyte survival - Improving the recruitment and homing of stem cells - Improve the efficiency of stem cell mediated repair of the damaged tissue

- 126 - Upon AMI, activated cytokines like granulocyte-stimulating factor (G-CSF) bind to the cytokine receptor on cardiomyocytes and activate the Jak/STAT3 signaling pathway. Activation of this pathway is known for its cardioprotective effects via the activation of pro-survival proteins like Akt and ERK. Ways to potentiate this pathway after an ischemic event could rescue cardiomyocytes and therefore result in a reduction in infarct size. Previously it was shown that miR-19 might protect cardiomyocytes during hypoxia through a yet unknown mechanism. Using a bioinformatic target prediction approach, we identified potential bindingsites for miR-19 in the 3’UTRs of suppressor of cytokine signaling (SOCS)1 and SOCS3, both known inhibitors of Jak/STAT3 signaling. We were able to demonstrate that in a mouse model of myocardial infarction (MI) Jak/STAT3 activity increases which corresponds to an increase in both SOCS1 and SOCS3. A set of in vitro and in vivo experiments indeed indicate that miR-19 has a protective effect under hypoxic conditions through the activation of Jak/STAT3. We have now created cardiomyocyte-specific transgenic mice to establish whether an increase in miR-19 leads to an increase in myocyte survival and improvement in cardiac function after MI. Just recently we showed that systemic delivery of a mimic can serve to therapeutically increase the level of a microRNA (Montgomery et al. 2014 EMBO Mol Med). These microRNA mimics harbour chemical modifications that improve their stability and cellular uptake, without interfering with its miRNA function. However, systemic delivery can also result in uptake by tissues that do not normally express the miRNA, resulting in potential off-target effects in unrelated cell types. To explore localized delivery techniques to enhance cardiac exposure we recently initiated a collaboration with Technical University of Eindhoven and the UMCU to explore the use of a supramolecular hydrogel to deliver miRNA therapeutics to the heart. Recently, these groups showed that a pH-responsive supramolecular hydrogel, can be used to effectively deliver drugs percutaneously to the infarcted porcine myocardium via a minimally invasive intramyocardial injection catheter. To enhance pharmacological activity while reducing the risk for unwanted toxicities, we will formulate miR-19 mimic in the UPy-hydrogel to study whether hydrogel-based delivery of miRNA therapeutics enhances the efficacy of miRNA therapeutics and whether localized cardiac delivery of miR-19 mimic improves cardiac regeneration in both small and large animal models of ischemic injury.

Another intriguing process for heart repair is based on the interaction between the chemokine stromal cell-derived factor (SDF)1 and its receptor C-X-C chemokine receptor type (CXCR)4. In response to tissue damage, SDF1 binding to CXCR4 is known to counteract apoptosis and enhance stem cell recruitment into a target area for subsequent tissue repair. Also in the heart SDF1 is a major chemokine which release is significantly upregulated after cardiac injury, including MI. In cardiomyocytes SDF1 can activate several pro-survival pathways like JAK/STAT, PI3K/Akt and ERK1/2, while, at the same time, it attracts CXCR4+ stem cells, like bone marrow derived stem cells (BMCs), towards the infarcted region in an SDF-1 dependent manner. Our lab is interesting in identifying mechanisms by which we can increase SDF-1/CXCR4 signaling in the ischemic myocardium. We are exploring the effect of local delivery of a non-cleavable form of SDF1. At the same time we are performing experiments to further study this pathway in the heart in more detail and we initiated screening efforts to identify microRNAs that can modulate this pathway via the regulation of SDF1, CXCR4 or both.

While the adult mammalian heart is essentially incapable of renewal after injury it does have some regenerative potential. While the origin of the key contributing cells is highly debated, c-kit cardiac progenitor cells have been a source of intense investigation. Recently, the group of Jeff Molkentin generated mice in which they targeted the c-kit locus with complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein in mice and then bred them with reporter lines. These data showed that endogenous c-kit cells did produce new cardiomyocytes within the heart, albeit at a much lower level than endothelial cells. Dr Molkentin kindly agreed to send us these mice, and in collaboration with the van Oudenaarden group we will attempt to define the gene expression patterns in these different cardiac c-kit positive cell populations and explore ways to stimulate this resident source of cardiac stem cells to differentiate into cardiomyocytes. Since it is also still unknown whether these c-kit cells divide asymmetrically, i.e., self-renew, or whether the labeled progeny derive from activation of a single or several stem cells, we will cross these mice to the R26R- confetti mice from the Clevers lab which should help answer these questions.

Based on the work from the Clevers lab we recently began to explore the involvement of Wnt signaling in heart injury. The Wnt target genes Lgr5, Lgr6, TNFRSF19 and Olmf4 (which mark specific stem cells in intestine, stomach and skin) are not expressed in the adult homeostatic heart. However, our own data indicate that ischemic injury in the heart activates Wnt signalling and transcription of target

- 127 - genes, like Lgr5 and Olfm4. We therefore speculate that heart injury induces the expression of these Wnt target genes in the presumptive progenitor population. Importantly, this hypothesis is not without precedent as other tissues such as liver and pancreas also do not express detectable levels of Lgr5. However, upon liver or pancreas injury Wnt signalling increases and Lgr5 is expressed. Using several lacZ reporters from multiple stem cell markers present in the Clevers lab, we are now collaboratively performing lineage tracing experiments to explore whether we can indeed define a new cardiac population of progenitors that is induced upon heart injury.

Societal relevance and societal impact (2008-2014)

Ischemic heart disease (IHD) is a modern world epidemic for which there currently are no effective therapies that could stop -or even reverse- disease progression with the exception of heart transplantation or assist devices. Unfortunately, these treatment options are only available to a minute fraction of the population in need of treatment due to donor scarcity, and are accompanied by incredibly high costs. Given the global burden of IHD and its increasing prevalence, the development of novel regenerative approaches is of the utmost importance. Since efforts to deliver exogenous stem cells have been disappointing so far, boosting endogenously present mechanisms might be a far more feasible way to go. Although there are many ways we could potentially stimulate these mechanisms, therapeutic manipulation of microRNAs has gained a lot of attention. Our lab has an ongoing sponsored research agreement with miRagen Therapeutics, a biotechnology company focused on the development of microRNA based therapies, of which I am a founder and for which I currently serve as special advisor. The agreement between the KNAW and miRagen is based on first right of refusal on any microRNA related IP that comes out of our lab. The enthusiasm for developing these new therapeutics is further underscored by the $352M partnership between miRagen and Servier for Research development and commercialization of miRNA-targeting drugs for cardiovascular disease (http://www.miragentherapeutics.com/4/News/).

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Utrecht, October 2014

Hubrecht Institute Uppsalalaan 8 3584 CT Utrecht +31 (0)30 212 18 00 www.hubrecht.eu

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