Stem Cells and Development

José  Belo  - 2801

José António Belo

Principal Investigator

CV

Location:

CEDOC
Campus Sant'Ana
Pólo de Investigação, NMS, UNL
Rua Câmara Pestana, nº 6
1150-082 Lisboa, Portugal

Phone: (+351) 218 803 101
E-mail: jose.belo(at)nms.unl.pt

mCcbe1_Ab

The current focus of our research lie in the fields of Stem Cell and Developmental Biology, mainly on cardiogenesis and Left-Right asymmetry. Since long ago we have been using ES cells, first as a tool for the generation of animal models of human diseases and recently as a tool for studying mechanisms of differentiation/regeneration.
We use mouse and human Embryonic Stem Cells, mouse, and chick embryos as model systems using the most up to date technologies in molecular embryology. Those include generation and analysis of Knock-Outs (KO) and transgenic mouse lines, CRISPR/CAS genetic manipulation as well as iPS cell generation and Stem Cell differentiation.

We have been involved in the study the establishment of the body axis of the early vertebrate embryo, in particularly the molecular pathways involved in the Anterior-Posterior (A-P) and Left-Right (L-R) visceral body asymmetry. In these fields we have contributed extensively with our work on Cerberus-like 1 (Cerl-1; AVE and A-P patterning) and on Cerberus-like 2 (Cerl-2; L-R patterning).

In the recent years we confirmed that Cerl2 is a key molecule for the establishment and termination of Nodal asymmetric activity in the mouse node (Inácio et al., 2013) and that Cerl2 is the major target of Pkd2-mediated signaling in crown cells cilia (Yoshiba et al., 2012). These results define Cerl2 as a crucial molecule in the control of the L-R axis.

As Cerl2 KO mice also display a unique phenotype characterized by an embryonic onset cardiac hyperplasia, this study lead us to a new and strong focus, which is the induction and control of cardiogenesis.

In a recent screen for genes with cardiogenic potential, we have identified among others the high profile gene Ccbe1, now under investigation by us and other labs around the world. Using mouse and human ES/iPS cells we are studying the role of Cerl2 and Ccbe1 in cardiogenesis.

Main accomplishments:
Cer1_GFP• Contribution to the understanding of the role the Anterior Visceral endoderm and of Cerberus1 and Cerberus/Dan family of secreted factors in the specification of vertebrate anterior embryonic structures.

• To confirm that Cerl2 is a key molecule for the establishment and termination of Nodal asymmetric activity in the mouse node (Inácio et al., 2013) and that Cerl2 is the major target of Pkd2-mediated signaling in crown cells (Yoshiba et al., 2012). These results define Cerl2 as a crucial molecule in the control of the L-R axis Cerberus like 2 (Cerl2) is a member of the Cer/Dan family, and has been show to antagonize signals from the TGF-beta superfamily by binding to, and thus inhibiting Nodal signals. Accordingly, Cerl2 knockout mice show a wide range of laterality and cardiac defects (Marques 2004).

• To demonstrate that enlargement of the Left Ventricule myocardial walls and interventricular septum (IVS) in Cerl2 null mutants without LD is caused by cardiomyocyte hyperplasia. Our results also indicate that the Nodal/pSmad2 signaling cascade is sustained in the absence of Cerl2, providing the first evidence of the role of Cerl2 during cardiac development.

• To perform a differential screen for early cardiogenic controlling genes (Bento et al., 2011). One of them, Ccbe1 now under intensive investigation. Ccbe1 loss-of-function in mice leads to prenatal death due to defective lymphatic vasculature and to cardiac defects as well. In humans, mutation in CCBE1 has been associated with Hennekam syndrome, a disorder characterized by abnormal lymphatic system development where some patients present as well congenital heart.

• To develop the use of ES cells beyond the current technology to generate genetically targeted mouse lines. We have started the use of mouse and human ES cell to study the control of ES cell differentiation towards cardiogenic tissue using Ccbe1 as a tool to enrich for the generation of ESC-derived cardiac progenitors and ultimately for the formation of cardiomyocytes.

• To develop and characterize novel natural and synthetic polymeric coatings for stem cell research and to validate their suitable use for mouse ESC culture.

LBG_ES

Selected Projects
Cerl2_node• As project leader

PTDC/BIM-MED/3363/2014 (2016/2019). CardioProD: Control of cardiomyocyte proliferation in disease and regenerative medicine

HMSP-ICT/0039/2013 (2014/2017) – CardioRegen: Integrative studies on the role of Ccbe1 in cardiogenesis: from the embryo to scalable multipronged generation of hESC-derived myocardial cells for heart tissue repair.

PTDC/SAU-ENB/121095/2010 - "Role of the novel ccbe1 growth factor in the generation of cardiac tissue from ES cells".

PTDC/SAU-BID/114902/2009 - "Role of the novel gene Ccbe in heart induction and organogenesis".

PTDC/SAU-GMG/101890/2008 - "Study of heart formation at molecular terms to understand congenital heart malformations".

• As project co-leader & collaborator

PAC/2015 (2016/2019). NETDIAMOND: NEw Targets in DIAstolic heart failure: from coMOrbidities to persoNalizeD medicine.

MITP-TB/ECE/0013/2013 (2014/2016) – CARDIOSTEM- Engineered cardiac tissues and stem cell-based therapies for cardiovascular applications. Principal Investigator: Lino Ferreira (CNC/Univ. Coimbra); Participating institutions: Univ. Coimbra, Univ. Técnica de Lisboa, Univ. Nova Lisboa, Univ. Lisboa.

PTDC/BIA-GEN/116519/2010 - "The impact of alternative splicing on structure and function of molecular networks during stem cell differentiation".

POCI/BIO/62476/2004 - “Viral Nanoparticles for Molecular and Gene Therapy”.

POCTI/ BIA-BCM/58677 /2004 - "Spatial-temporal pattern of expression and regulation of Matrix Gla Protein (MGP) during early development in X. laevis".
Cerl2_Nodal

Selected Publications

Doppler

  • Ana Rubina Perestrelo AR, Ana Grenha, Ana M. Rosa da Costa, José António Belo (2014). Locust Bean Gum as an alternative polymeric coat- ing for embryonic stem cell culture, Materials Science & Engineering C, DOI: 10.1016/j.msec.2014.04.022
  • Vinhas M, Araújo AC, Ribeiro S, Brás Rosário L and Belo JA (2013). Transthoracic echocardiography reference values in juvenile and adult 129/Sv mice. Cardiovascular Ultrasound 11:12 (doi:10.1186/1476-7120-11-12).
  • Inácio JM, Marques S, Nakamura T, Shinohara K, Meno C, Hamada H, and Belo JA (2013). The Dynamic Right-to-Left Translocation of Cerl2 is Involved in the Regulation and Termination of Nodal Activity in the Mouse Node. PLoS ONE 8(3): e 60406. doi:10.1371/journal.pone.0060406
  • Ana Rubina Perestrelo, Fouzi Mouffouk, Ana M. Rosa da Costa and José António Belo (2013). Novel triblock copolymer nanofiber system as an alternative support for embryonic stem cells growth and pluripotency. T. Eng. Reg. Med. Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1838.
  • Nakamura T, Saito D, Kawasumi A, Shinohara K, Asai Y, Takaoka K, Dong F, Takamatsu A, Belo JA, Mochizuki A, Hamada H. (2012) Fluid flow and interlinked feedback loops establish left-right asymmetric decay of Cerl2 mRNA. Nat. Commun. 3:1322. [doi: 10.1038/ncomms2319].
  • Satoko Yoshiba, Hidetaka Shiratori, Ivana Y. Kuo, Aiko Kawasumi, Kyosuke Shinohara, Shigenori Nonaka, Yasuko Asai, Genta Sasaki, Jose António Belo, Hiroshi Sasaki, Junichi Nakai, Bernd Dworniczak, Barbara E. Ehrlich, Petra Pennekamp and Hiroshi Hamada (2012) Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2.Science 338(6104):226-31.
  • Facucho-Oliveira, J., Bento, M. and Belo, J.A. (2011) Ccbe1 expression marks the cardiac and lymphatic progenitor lineages during early stages of mouse development. Int. J. Dev. Biol. 55 (10-11-12):1007-1014.
  • Chi L, Saarela U, Railo A, Prunskaite-Hyyryläinen R, Skovorodkin I, Anthony S, Katsu K, Liu Y, Shan J, Salgueiro AM, Belo JA, Davies J, Yokouchi Y, Vainio SJ.(2011) A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development.PLoS One 6(11):e27676. PMID: 22114682.
  • Margaret Bento, Elizabeth Correia, Ana T. Tavares, Jorg Becker and José A. Belo. (2011) Identification of differentially expressed genes in the heart precursor cells of the chick embryo. Gene Expr. Patterns 11, 437-447.
  • Aiko Kawasumi, Tetsuya Nakamura, Naomi Iwai, Kenta Yashiro, Yukio Saijoh, Jose António Belo, Hidetaka Shiratori, and Hiroshi Hamada. (2011) Left-right asymmetry of Nodal activity in the node is translated into left-right asymmetry in the lateral plate of mouse embryos. Dev Biol. 353(2):321-30.
  • Lisa Gonçalves*, Mario Filipe*, Sara Marques, Jorg Becker, José A. Belo (2011) Isolation and study of novel head-inducing genes expressed in the AVE reveals a role for ADTK1 in mouse development. Int. J. Dev. Biol. 55:281-295. (* equal authors).
  • José A. Belo, Ana C. Silva, Ana C. Borges, Mário Filipe, Margaret Bento, Lisa Gonçalves, Marta Vitorino, Ana M. Salgueiro, Vera Teixeira, Ana T. Tavares & Sara Marques (2009) Generating asymmetries in the early vertebrate embryo: the role of the Cerberus-like family. Int. J. Dev. Biol. 53(8-10):1399-407.
  • Giovanna L. Liguori*, Ana C. Borges*, Daniela D’Andrea, Annamaria Liguoro, Lisa Gonçalves, Ana M. Salgueiro, M. Graziella Persico & José A. Belo. (2008) Cripto independent nodal signalling promotes positioning of the A-P axis in the early mouse embryo. Dev Biol.15;315(2):280-9 (* equal authors).
  • Kimura-Yoshida, C., Nakano, H., Okamura, D., Nakao, K., Yonemura, S., Belo, J.A., Aizawa, S., Matsui, Y. and Matsuo, I. (2005) Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm. Dev. Cell 9 : 639-650
  • Marques, S., Borges, A.C., Silva, A.C., Freitas, S., Cordenonsi, M., and Belo, J.A. (2004) The activity of the Nodal antagonist Cerl-2 in the mouse node is required for correct L/R body axis. Genes & Development 18 : 2342-2347
  • Silva, A., Filipe, M., Kuerner, K., Steinbeisser, H. and Belo, J.A. (2003) Endogenous Cerberus activity is required for anterior head induction in Xenopus. Development 130 : 4943-4953
  • Belo, J.A., Bachiller ,D., Agius, E., Kemp, C., Borges, A.C., Marques, S., Piccolo, S. and De Robertis, E.M. (2000) Cerberus-like is a BMP4 and Nodal antagonist not essential for mouse development. Genesis 26 : 259-264
  • Bachiller, D., Klingensmith, J., Kemp, C., Belo, J.A., Anderson, R.M., May, S.R., McMahon, J.A., McMahon, A.P., Harland, R.M., Rossant, J. and De Robertis, E.M. (2000) The organizer secreted factors chordin and noggin are required for forebrain development in the mouse. Nature 403 : 658-661
  • Belo, J.A., Leyns, L., Yamada, G. and De Robertis, E.M. (1998) The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants. Mech. Dev. (issue cover) 72 : 15-25
  • Belo, J.A., Bouwmeester, T., Leyns, L., Kertesz, N., Gallo, M., Folletie, M., and De Robertis, E.M. (1997) Cerberus-like is a secreted factor with neuralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. (issue cover) 68 : 45-57

Books

    • Belo, J.A. and Vieira, P (2011) Ratinhos transgénicos. (Transgenic mice). In Videira A. (ed.) “Engenharia genética – Princípios e aplicações”.2.ª Edição, Lidel. Lisboa, pp.135-150. (Portuguese).

cCer_GFP

• Herbert Steinbeisser, University of Heidelberg, Heidelber, Germany – Mcer-l, Ccar and Xcer promotor studies in Xenopus embryos in order to access the mechanisms of forebrain induction by the anterior endoderm. Role of novel genes in the control of the Wnt PCP pathway.

• Hiroshi Hamada, Osaka University, Osaka, Japan – Studies on the role of the Cerl-2 gene in the establishment of early Left/Right asymmetries in the mouse embryo.

• Ibrahim Domian, Harvard Medical School, Cambridge, USA – Use of mouse ES cells to study the control of ES cell differentiation towards cardiogenic tissue

• Roger Pedersen, Cambridge University, UK – Study of the role of novel genes using hES cells.

Nanofibers_ES