Homeoproteins and Cell Plasticity
Principal Investigators: Alain JOLIOT, DR2 Cnrs & Sophie VRIZ Pr. Paris Diderot
Alain Joliot and Sophie Vriz Teams joined recently to conduct a project focus on the analysis of homeoprotein function in regeneration.
Regeneration and Progenitor Cell Recruitment in Adult
Our team has a long-standing interest in zebrafish biology that originates from the study of adult fin regeneration, an attractive model that recapitulates many developmental processes, including plasticity (dedifferentiation), proliferation and patterning (during regrowth). Importantly, regeneration also addresses several key issues of regenerative medicine. Following the loss of an appendage, epimorphic regeneration in amphibians and fish provides relevant models of integrated progenitor cell recruitment, where stump cells of different lineages dedifferentiate to acquire a progenitor identity in response to the injury. Subsequently, they accumulate at the damaged surface to form a mass of proliferating cells: the blastema, which depends on the dedifferentiation of cells that return to progenitor cell identity and on their migration underneath the wound epithelium. The missing link between amputation (or injury) and progenitor cell proliferation is the nature of the signals that are essential to push differentiated cells into a progenitor cell state. Our studies recently focussed on early signals involved in regeneration of the adult zebrafish caudal fin. Converging arguments support the idea that progenitor recruitment and proliferation require an interaction between epidermis and the underlying mesenchyme. We showed that reactive oxygen species (ROS) and apoptosis signalling in the stump epidermis play a major role in mesenchymal progenitor cell mobilization (Gauron et al. (2013) Sci Rep 3:2084). This finding suggests that ROS signalling may be one of the cues for information transfer from the stump epidermis to the underneath mesenchyme, but questions how this information spreads in the damaged tissue and raises the question of the nature of the cues (extrinsic or intrinsic) at stake. Among them, homeoproteins are obvious candidates, due to their central role in pattern formation during development.
In vivo transduction of CRE-SecPenetratin: In vivo delivery of a CRE-SecPenetratin recombinant protein by retina injection induces massive recombination events (green) throughout the retina layers.
Engrailed photoactivation during Development
Mechanisms of Homeoprotein Action
Homeoproteins are a class of transcription factors defined by the structure of their DNA-binding domain, the homeodomain. They are key regulators of multiple developmental processes and exhibit additional functions in the adult. Homeoproteins are unique in their ability to act through intracrine or paracrine mode of action. The latter action relies on their transfer between cells, involving unconventional secretion and internalisation processes. Our goal is to decipher the molecular and cellular mechanisms of the transfer, with a specific interest in the addressing of these nuclear proteins towards the plasma membrane and in their translocation across the membrane, that can occur in both directions (in and out) and involve direct interactions with phospholipids and sugars.
Homeoprotein paracrine action is a key component of homeoprotein function, from drosophila to mouse, and is antagonized by the addition of extracellular blocking antibodies against homeoproteins. Base on this strategy, originally developed in the zebrafish embryo our team has have recently identified a paracrine action of Engrailed proteins in the formation of diencephalon/midbrain boundary in zebrafish. We will use this model as a paradigm to design and validate new strategies that specifically target homeoprotein paracrine action. They will rely on the spatio-temporal photo control of protein activity (see below) and the use of modulators of homeoprotein intercellular transfer, acting on secretion or internalisation.
Role of Homeoproteins During Regeneration
The identification of homeoprotein candidates in regeneration will rely on two complementary strategies. In a targeted strategy, homeoproteins already associated to the regeneration process (e.g. msx) or linked to the signalling pathways that control regeneration (e. g. Oct1 and ROS signalling) will be analysed, in vivo during regeneration by gain or loss of function approaches, and ex vivo to specifically investigate their intercellular transfer behaviour (secretion and internalisation). The second unbiased strategy will be based on transcriptomic and proteomic data from the literature to extract the expression profile of homeoproteins, which will be further refined in the team, due to our specific interest in the early steps of regeneration. The selected candidates will be processed as for the targeted strategy.
Protein Delivery and Photocontrol of Protein Activity
Non-invasive tools to study cell physiology in vivo are still too few. We thus decided to design new tools in collaboration with two scientists of Ecole Normale Supérieure, Ludovic Jullien, chemist (UMR8640, PASTEUR) and David Bensimon, physicist (UMR8550, LPS). We developed photoactivatable molecules and their application to fundamental biological processes. We have now reached the single cell level in vivo and are able to manipulate protein activity (specificaly caspase, ROS production and homeoprotein activation). These Tools are combined to biosensors to decipher signalling spreading in vivo.
Direct delivery of proteins from the extracellular space is an alternative strategy developed in the team to control protein activity, which has been successfully achieved upon fusion of the protein cargo with Cell Penetrating Peptides (CPPs). The respective efficacies and scellular tropism of different types of CPPs are analysed in dedicated in vivo mouse or zebrafish models.
Homeoprotein Internalization: Extracellular FITC-labelled Engrailed homeoprotein (green) accumulates in cytosolic and nuclear compartments of live CHO cells.
Selected Publications 2008-2017
- Feng, Z., Nam, S., Hamouri, F., Aujard, I., Ducos, B., Vriz, S., Volovitch, M., Jullien, L., Lin, S., Weiss, S. & Bensimon, D. (2017), Optical Control of Tumor Induction in the Zebrafish. Sci Rep 7, 9195.
- Meda, F., Rampon, C., Dupont, E., Gauron, C., Mourton, A., Queguiner, I., Thauvin, M., Volovitch, M., Joliot, A. & Vriz, S. (2017b), Nerves, H2O2 and Shh: Three players in the game of regeneration. Semin. Cell Dev. Biol.
- Meda, F., Joliot, A. & Vriz, S. (2017), Nerves and hydrogen peroxide: how old enemies become new friends. Neural Regen Res 12, 568–569.
- Chen, Y., Ma, J., Lu, W., Tian, M., Thauvin, M., Yuan, C., Volovitch, M., Wang, Q., Holst, J., Liu, M., Vriz, S., Ye, S., Wang, L. & Li, D. (2016) Heritable expansion of the genetic code in mouse and zebrafish. Cell Res. Dec 9. (Letter to the Editor, Cell Research 27, 294-297- February 2017).
- Gauron, C., Meda, F., Dupont, E., Albadri, S., Quenech’Du, N., Ipendey, E., Volovitch, M., Del Bene, F., Joliot, A., Rampon, C. & Vriz, S. (2016), Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development. Dev. Biol. pii: S0012-1606 (15)30311-0.
- Plamont, M.-A., Billon-Denis, E., Maurin, S., Gauron, C., Pimenta, F.M., Specht, C.G., Shi, J., Quérard, J., Pan, B., Rossignol, J., Morellet, N., Volovitch, M., Lescop, E., Chen, Y., Triller, A., Vriz, S., Le Saux, T., Jullien, L. & Gautier, A. (2016), Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo. Proc. Natl. Acad. Sci. U.S.A. 113, 497–502.
- Meda, F., Gauron, C., Rampon, C., Teillon, J., Volovitch, M. & Vriz, S. (2016), Nerves Control Redox Levels in Mature Tissues Through Schwann Cells and Hedgehog Signaling. Antioxid. Redox Signal. 24, 299–311.
- Dupont E., Prochiant, A. & Joliot A. (2015), Penetratin Story: An Overview. Methods Mol. Biol. 1324, 29–37.
- Rampon C., Gauron C., Lin T., Meda F., Dupont E., Cosson A., Ipendey E., Frerot A., Aujard I., Le Saux T., Bensimon D., Jullien L., Volovitch M., Vriz S. & Joliot, A. (2015), Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain. Development 142, 1840–1849.
- Querard J., Markus T.-Z., Plamont M.-A., Gauron C., Wang P., Espagne A., Volovitch M., Vriz S., Croquette V., Gautier A., Le Saux T. & Jullien L. (2015), Photoswitching Kinetics and Phase-Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging. Angew. Chem. Int. Ed. Engl. 54, 2633–2637.
- Gautier A., Gauron C., Volovitch M., Bensimon D., Jullien L. & Vriz S. (2014), How to control proteins with light in living systems. Nat. Chem. Biol. 10, 533–541.
- Rampon C., Gauron C., Meda F., Volovitch M. & Vriz S. (2014), Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration, Purinergic Signal, Aug 2.
- Vriz S., Reiter S. & Galliot B. (2014), Apoptosis : a program to regenerate. Curr Topics in Dev Biol. 108, 121–151.
- Gauron C., Rampon C., Bouzaffour M., Ipendey E., Teillon J., Volovitch M. & Vriz S. (2013), Sustained production of ROS triggers compensatory proliferation and is required for regeneration to proceed. Scientific Reports 3, 2084.
- Feng Z., Zhang W., Xu L, Gauron C., Vriz S., Volovitch M., Jullien L., Weiss S. & Bensimon D. (2013), Optical control and study of biological processes at the single cell level in a live organism. Reports on Progress in Physics. 76(7): 072601.
- Fournier L., Gauron C., Xu L., Aujard I., Le Saux T., Gagey-Eilstein N., Maurin S., Dubruille S., Baudin J.-B., Bensimon D., Volovitch M., Vriz S. & Jullien L. (2013), A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation. ACS Chem. Biol. May7.
- Spatazza J., Lee H.H., Di Nardo A.A., Tibaldi L., Joliot A., Hensch T.K. & Prochiantz A. (2013), Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity. Cell Report, Volume 3, Issue 6,1815-1823.
- Spatazza J., Di Lullo E., Joliot A., Dupont E., Moya K.L. & Prochiantz A. (2013), Homeoprotein signaling in development, health, and disease: a shaking of dogmas offers challenges and promises from bench to bed. Pharmacol Rev. Jan 8;65(1):90-104.
- Sagan S., Burlina F., Alves I.D., Bechara C., Dupont E. & Joliot A. (2013), Homeoproteins and homeoprotein-derived peptides: going in and out. Curr Pharm Des. 19(16):2851-62.
- Xu L., Feng Z., Sinha D., Ebenstein Y., Gauron C., Le Saux T., Lin S., Weiss S., Vriz S., Jullien L. & Bensimon D. (2012), Spatio-temporal manipulation of retinoic acid activity in zebrafish hindbrain development via photo-isomerization. Development, 139(18):3355-62.
- Di Lullo E., Haton C., Le Poupon C., Volovitch M., Joliot A., Thomas J.-L. & Prochiantz A. (2011), Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube. Development, 138, 4991-5001.
- Layalle S., Volovitch M., Mugat B., Bonneaud N., Parmentier M.-L., Prochiantz A., Joliot* A. & Maschat F.* (2011), Engrailed homeoprotein acts as a signaling molecule in the developing fly. Development, 138, 2315-2323.
- Hoang T.Q., Rampon C., Freyssinet J.M., Vriz S. & Kerbiriou-Nabias D. (2011), A method to assess the migration properties of cell-derived microparticles within a living tissue. BBA-GEN SUBJECTS 1810(9):863-6.
- Bouzzafour M., Rampon C., Ramaugé M., Courtin F. & Vriz S. (2010), Differential effect of thyroid hormones during regeneration in zebrafish. Gen Comp Endocrinol, 2010 Aug 1;168(1):88-94.
- Sinha D., Neveu P., Gagey N., Aujard I., Le Saux T., Rampon C., Gauron C., Kawakami K., Leucht C., Bally-Cuif L., Volovitch M., Bensimon D., Jullien L. & Vriz S. (2010), Photo-activation of the CreERT2 recombinase conditional site-specific recombination with hogh spatio-temporal resolution. Zebrafish, Jun;7(2):199-204.
- Sinha D., Neveu P., Gagey N., Aujard I., Benbrahim-Bouzidi C., Le Saux T., Rampon C., Gauron C., Goetz B., Dubruille S., Baaden M., Volovitch M., Bensimon D., Vriz S. & Jullien L. (2010), Photocontrol of protein activity in cultured cells and zebrafish with one- and two-photon. ChemBioChem, 11(5) : 653-663.
- Wizenmann A., Brunet I., Lam J.S.Y., Sonnier L., Beurdeley M., Zarbalis K., Weisenhorn-Vogt D., Weinl C., Dwivedy A., Joliot A., Wurst W., Holt C. & Prochiantz A. (2009), Extracellular engrailed participates in the topographic guidance of retinal axons in vivo. Neuron 64, 355-366.
- Gitton Y., Tibaldi L., Dupont E., Levi G. & Joliot A. (2009), Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues. BMC Biotechnol 9, 40.
- Bouzaffour M., Dufourcq P., Lecaudey V., Haas P. & Vriz S. (2009), Fgf and Sdf-1 pathways interact during zebrafish fin regeneration. PLoS ONE, 4(6): e5824.
- Neveu P., Aujard I., Benbrahim C., Le Saux T., Allemand J.F., Vriz S., Bensimon D. & Julien L. (2008), A caged retinoic acid for use with one and two photon excitation in zebrafish embryos. Angewandte Chemie Int Ed Engl, 47, 3744-46.
Joliot Alain, DR2 CNRS
Vriz Sophie, Professor Paris-Diderot
Rampon Christine, MDC Paris-Diderot
Postdoctoral fellows & PhD Students:
Mourton Aurélien, PhD student
Amblard Irene, PhD student
Dupont Edmond, IR1 CNRS
Queguiner Isabelle, AI CDF
Thauvin Marion, CDD AI CDF
Lebled Valérie, TCN CNRS