A central question in developmental biology is how cells decide which differentiation paths to follow, during the generation of tissues and organs throughout embryonic development. Although genetic information is identical in all cells, the genetic space that is available during each cell-fate decision varies according to the cell’s developmental history and its position in the embryo. In our laboratory, we are trying to elucidate i) the rules that delimit the genetic space available to undifferentiated cells in the embryo, and, ii) how cells explore the available space during cell-fate decision processes. Two different cellular models are used to investigate these questions. In the first, we are using mouse embryonic stem cells (ESCs) to study the molecular mechanisms regulating pluripotency and lineage specification (“two sides of the same coin”). In the second, we are using the embryonic neural retina to study how retinal progenitors acquire their multipotent features and generate the variety of neurons that compose the adult eye. In both cases, our main aim is to understand the gene regulatory networks that underlie the broad lineage potential of stem/progenitor cells and, concomitantly, govern the decision processes that these cells undertake to differentiate along various lineages, to generate correctly patterned tissues and organs.

 

Domingos Henrique, PhD – Auxiliary Investigator, FMUL
PhD (1991) University of Lisbon
Post-doctoral research at NIMR and ICRF (UK), and at the Institut d’Embryologie Cellulaire et Moléculaire (France)
Investigator at the Faculty of Medicine, University of Lisbon

 

 

 

Stem cells are characterized by their self-renewal capacity and competence for multilineage differentiation (pluripotenciality). They are important not only for the development of the organism but also for its sustained maintenance and growth. Embryonic stem (ES) cells constitute one type of stem cell derived from the inner cell mass (ICM) of the blastocyst that can be maintained in vitro under very specific culture conditions. ES cells have the capacity to generate all embryonic tissues and are therefore a promising resource for the next-generation of cellular therapies, in the emerging area of regenerative medicine. This area is going through a “revolutionary” period, after the report in 2006 by Takahashi and Yamanaka that ectopic expression of only four genes (Oct4, Sox2, Klf4 and c-Myc) is able to reprogramme mouse embryonic fibroblasts into ES-like pluripotent cells, named “induced Pluripotent Stem”– iPS cells.

Since then, several papers have reported the in vitro reprogramming of various somatic cell types, including differentiated adult cells, a finding with obvious therapeutic implications, in particular concerning the use of iPS cells for in vitro disease modelling, through the generation of patient-specific stem cells, and for the development of cellular replacement therapies. However, the phenomenon of gene-induced reprogramming is still very inneficient and remains a “black box” where little is known about the nature and sequence of events along the path to recreate a “stemness” state. This is a major obstacle to the widespread use of iPS cells in regenerative medicine and emphasizes the need to reach a more extensive comprehension of the genetic and biochemical pathways underlying reprogramming of the “stemness” state in somatic cells. An important step in this direction was recently provided by the emergence of a new concept of “stemness” as a “uninstructed ground state” that is intrinsically self-maintaining if protected from inductive differentiation stimuli. Application of this concept to iPS generation might lead to more rational strategies to induce “stemness” in somatic cells and, actually, resulted already in efficient conversion of neural stem cells into fully reprogrammed iPS cells.
The regulatory circuitry underlying “stemness” has also been extensively dissected and a consensual view is that the NOS network of transcription factors (including Nanog, Oct4 and Sox2) function together at the top of this circuitry. Still, how “stemness” emerges from the integrated activity of this network is far from understood, despite the extensive characterization of interacting partners, downstream targets and auto/cross-regulations between the 3 factors. The challenge is therefore to understand the dynamic behaviour of the NOS network and the emergent properties that underlie the establishment of a “stemness” state.

Our starting point in this project was the reported heterogeneity of Nanog expression levels in both ES cells and ICM cells from the blastocyst, which indicates a possible oscillatory behavior of Nanog gene’s activity. This is supported by other findings showing that Nanog expression levels indeed fluctuate in individual ES cells. Our lab at IMM has been working on another regulatory circuitry that also shows oscillatory behaviour (the Notch network) and has been developing tools to follow gene expression oscillations in live embryos and ES cells. We were therefore primed to notice the functional implications of Nanog oscillations concerning the nature of the “stemness” state. We are also aware of the need to use quantitative and dynamic methods to monitor these oscillations, so that mathematical modelling tools can be used to extract new insights about the functional architecture of the NOS (and Notch) genetic circuitry. Our aim is therefore to employ a “systems biology” approach to understand this architecture, how it produces the observed fluctuations in Nanog levels and how these fluctuations might underlie the emergence of the dialectic state of “stemness”: a state in which individual stem cells are able to simultaneously manifest their self-renewal and pluripotency features while being “open for proposals”, i.e., ready to respond to omnipresent differentiationinducing signals.

Our lab at IMM has accumulated a good expertise in ES cell biology and we think that, as a team, we are in excellent position to address the question of how the NOS circuitry functions to generate the unique and fundamental cellular state of “stemness”.

 

 

The pioneering work of Ramon y Cajal provided the first intelligible description of the cellular complexity in the vertebrate nervous system, and framed the fundamental question of how neuronal identity is generated during development. One century later, it is well-established that the complex anatomical organization of the nervous system is anticipated by a cascade of molecular regionalization events in the neuroepithelium, starting at early stages of embryonic development and resulting in the delimitation of
distinct progenitor domains, each expressing unique combinations of transcription factors (TFs). Understanding how distinct neuronal types arise can, therefore, be reduced to the question of how such combinatorial gene expression mechanisms are established during development. Two main strategies seem to be used, the first involving progenitor intrinsic programs that regulate, in a cell-autonomous  manner, the sequence of neuronal cell types produced, and the second based on cell-to-cell communication between progenitors, as the main determinant of the resulting neuronal types.

The challenge is therefore to elucidate how the interplay between intrinsic and extrinsic factors determines the acquisition of unique neuronal differentiation programs. This will be crucial to understand how neuronal circuits assemble in the brain and how they control specific behaviors in the adult, a major challenge to “modern” neuroscience. In addition, this knowledge will be critical to design more rational strategies to produce specific neuronal types from ES cells or iPS cells, a key step in the development of regenerative therapies.

The spinal cord constitutes an excellent model to study the mechanisms underlying specification of neuronal identities in the developing CNS and, in this project, we propose to investigate the molecular strategies underlying the production of functionally distinct interneurons in a particular region of the spinal cord, the V2 domain. Our previous work has contributed to highlight some features of V2 neurogenesis, namely that it is coordinated by the action of extrinsic cues, with Notch signalling mediated by two different Dll ligands playing a pivotal role, and intrinsic factors, like the proneural bHLH genes Ascl1 and Ngn1/2. We now propose to characterize the main components of the V2 regulatory network and investigate how they interact functionally to specify the correct number and types of interneurons in the V2 domain. In the first part of the project, we shall address the function of the proneural bHLH genes Ascl1 and Ngn1/2 during the initial steps of V2 lineage development, in particular how they control the competence of V2 progenitors. We shall then address the role of the two Notch ligands in the process, Dll1 and Dll4, aiming to elucidate how they are integrated in the V2 regulatory network and what V2 cell fate decisions they might control. Finally, we propose a series of fatemapping experiments to reveal the order of expression of various TFs along the various steps of V2 neuronal development, aimed to identify candidate regulators upstream of the two Dll genes during V2 neurogenesis. We shall use this information as entry point to start dissecting the regulatory steps of the V2 network at which Dll4 participate, by defining the promoter regions responsible for its
expression in V2 cells and identifying which TFs (bHLH and others) bind directly to these regions and regulate Dll4 expression.
Altogether, this project is expected to contribute to a better knowledge of the V2 regulatory network and elucidate how intrinsic and extrinsic factors coordinate their activities during neuronal fate decisions. In the long-term aim, we expect this project might contribute to reach a more comprehensive view of the molecular logic underlying neuronal specification in the developing nervous system.

 

Arjun Raj, University of Pennsylvannia, USA

http://rajlab.seas.upenn.edu/

Ana Nunes, Faculdade de Ciências, UL, Portugal

http://physbio.cii.fc.ul.pt/PBS/people.html

Ana Pombo, MRC Clinical Sciences Centre, UK 

http://www.csc.mrc.ac.uk/Research/Groups/EDC/GenomeFunction/

Petr Svoboda, Institute of Molecular Genetics, Czech Republic

http://www.img.cas.cz/public/skupiny/Svoboda.html

Nicolas Daudet / Jonathan Gale, UCL Ear Institute (UCL School of Life and Medical Sciences), London, U.K.
http://www.ucl.ac.uk/ear/research/daudetlab
http://www.ucl.ac.uk/ear/research/profiles/gale

Johan Van Es, Hubrecht Institute, Utrecht, The Netherlands
http://www.hubrecht.eu/research/clevers/index.html

Participation on the annual programme for visiting students from various educational levels, organized by “Instituto de Medicina Molecular”, Lisbon, Portugal

Annual participation on Brain Awareness Week activities.

http://www.dana.org/brainweek/

Participation in several “science-to-the-public” AR events. http://ar.neuro.fchampalimaud.org/
http://ar.neuro.fchampalimaud.org/event/human-2-point-0

Participation in the project “Sciencecalifragilistic – a Journey through the scientific method”.

 

 

BioMedCentral Neuroscience, Neurology and Psychiatry Award 2012 for the article published in BMC Biology: "A novel reporter of notch signalling indicates regulated and random notch activation during vertebrate neurogenesis"

Best Poster 2012 for Catarina Ramos at the Sinal 2012 - 6th National Meeting on Cell Signalling, Braga, Portugal:  "The role of different Notch ligands in the control of spinal cord neurogenesis"

1st Prize Award ‘The Physics of Life’ contest, EMBO BMD2011 Workshop, May 2011, Oeiras, Portugal: "The Heart of a Brain"

ESTOOLS 1st Prize for Best Poster to Elsa Abranches at the ESTOOLS scientific symposium - Stem Cells in Biology and Disease, May 2010. Lisbon, Portugal: "From ES cells to Neurons: Dissecting Notch function during Neurogenesis"

Gulbenkian Prize of Science, with Isabel Palmeirim, David Ish-Horowicz and Olivier Pourquié (1997).

Generation and characterization of a novel mouse embryonic stem cell line with a dynamic reporter of Nanog expression
Abranches, E., Bekman, E., Henrique, D.
PLoS One 2013. 8(3): e59928

Glycogen synthase kinase-3 inhibition enhances translation of pluripotency-associated transcription factors to contribute to maintenance of mouse embryonic stem cell self-renewal
Sanchez-Ripoll, Y., Bone, H.K., Owen, T., Guedes, A.M., Abranches, E., Kumpfmueller, B., Spriggs, R.V., Henrique, D. and Welham, M.J.
PLoS One 2013. 8(4): e60148)

Stochastic single-gene autoregulation
Aquino T, Abranches E, Nunes A.
Phys Rev E Stat Nonlin Soft Matter Phys 2012 Jun. 85(6 Pt 1):061913.

A novel Reporter of Notch Signalling indicates regulated and random Notch Activation during Vertebrate Neurogenesis
Vilas-Boas, F., Fior, R., Swedlow, J.D., Storey, K.G., Henrique, D.
BMC Biology 2011. 9:58.

HES6-1 and HES6-2 function through different mechanisms during neuronal differentiation
Vilas-Boas, F., Henrique, D.
PLoS One 2010. 5(12):e15459.

Two Notch ligands, Dll1 and Jag1, are differently restricted in their range of action to control neurogenesis in the mammalian spinal cord
Ramos C, Rocha S, Gaspar C, Henrique, D.
PLoS One 2010. 24, 5(11):e15515.

A cross disciplinary approach to understanding neural stem cells in development and disease
Henrique, D., Bally-Cuif, L.
Development 2010. 137:1933-8.

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation
Schulz, H., Kolde, D., Adler, P., Konstantinos Anastassiadis, Michael Bader, Nathalie Billon, Hélène Boeuf, Pierre-Yves Bourillot, Frank Buchholz, Christian Dani, Xavier Doss, Lesley Forrester, Ruairi Friel, Murielle Gitton, Domingos Henrique, Jürgen Hescheler, Heinz Himmelbauer, Norbert Hübner, Efi Karantzali, Androniki Kretsovali, Sandra Lubitz, Laurent Pradier, Jüri Reimand, Alexandra Rolletschek, Agapios Sachinidis, Pierre Savatier, Francis Stewart, Mike P. Storm, Marina Trouillas, Jaak Vilo, Melanie J. Welham, Johannes Winkler, Anna Wobus and Antonis K. Hatzopoulos
PlosOne 2009. 4(9):e6804.

Neural Differentiation of Embryonic Stem Cells in vitro: a Road Map to Neurogenesis in the Embryo Abranches, E., Silva, M., Pradier, L., Schulz, H., Hummel, O., Henrique, D., Bekman, E.
PLos ONE 2009. 4(7):e6286

Notch-Off”: a perspective on the termination of Notch signalling
Fior, R, Henrique, D.