The mammalian cerebral cortex has been subject to a dramatic expansion in surface area along the evolutionary scale. This process is accompanied by the appearance of stereotyped folds and fissures, and is recapitulated during normal development in the majority of higher mammals, including carnivores and primates. The development of the cerebral cortex involves three major steps: production of neurons, migration of neurons along radial glia, and alignment of neurons into discrete layers. Recent studies in mice have shown that certain alterations in the rates of proliferation and cell death of cortical progenitors result in an overproduction of cells, and an artificial expansion and folding of the mouse cerebral cortex. However, virtually nothing is known about the mechanisms determining the formation of stereotyped folding patterns in the cerebral cortex of higher mammals, and even less is known about the mechanisms that choreograph the processes of increased proliferation, radial migration, and layering, that give rise to a gyrated cerebral cortex in those species. To this aim, we propose to use a combination of in vivo and in vitro approaches in both ferrets and mice to achieve three specific objectives: i) to unravel the changes occurring in cortical progenitors related to cortical expansion in a naturally expanded cerebral cortex ii) to understand the mechanisms that ensure the existence of a radial glial scaffold for neuron migration during cortical growth iii) to determine the mechanisms controlling that CR cells (which orchestrate neuronal migration and layer formation) are present throughout the marginal zone of the tangentially expanding cerebral cortex.

The neocortex decodes sensory information from the external world. Cortical neurons are organized in columns, each tuned to one specific sensory stimulus. A growing body of evidences suggests that the neocortex directly participates in associative learning too. A stimulus that is initially neutral to a mouse, like the deflection of a whisker, acquires the emotional value of another sensory stimulus, like a tail shock, when the two are presented together. After conditioning, the mouse freezes at the deflection of that whisker alone. This learning correlates with changes in the barrel cortex, the region that contains the somatotopic representation of whiskers: more neurons become responsive to the conditioned whisker, regardless of their column. In the wild, this mechanism could “let the animal know” what specific signal to look for in order to adopt the behavior that might save its life, like freezing at the sound of a rattlesnake. Still, the specific cortical circuits involved in this phenomenon remain largely unknown. I will combine behavioral studies with electrophysiological recordings to unveil them. I will use the technique of glutamate uncaging to map the neuronal connectivity in brain slices. I will be able to identify the connections that are changed by associative learning and describe the cellular mechanisms that changed them. I will also compare the ontogeny of associative learning with the maturation of cortical connections. Finally, the comparison of normal mice with models of mental retardation will further reveal the role of cortical networks in learning. The carrier development award will help me to establish this new line of research in my home country.

miRNAs play fundamental role in shaping gene expression. Yet, the mechanisms by which miRNAs control gene expression remain largely unknown. The miRNA-mediated repression complex was shown to localize to so-called P bodies, cytoplasmic foci containing non-translated mRNAs and degradation enzymes. Moreover, some of P body components were shown to play a role in miRNA pathway. Hence, thorough analysis of P body content and structural organization would help to elucidate the mechanisms of miRNA-directed repression. I propose to analyze the composition of P bodies and examine the role of newly identified P body components in the regulation of gene expression by miRNAs. First, I propose to isolate P bodies from mammalian cell culture using affinity tags fused to various known components of P bodies and analyze the content of the purified complexes by mass spectrometry and microarray analysis. Second, in parallel with the biochemical technique, I propose to do biocomputational search for novel component of P bodies. Third, the novel components of P bodies identified during this study will be analyzed for their role in miRNA pathway, using reporter system recapitulating miRNA-directed repression in mammalian cell culture. Fourth, based on the data about intermolecular interactions within P bodies, I will be able to determine distinct compartments within P bodies. The analysis of mRNA distribution between such compartments may lead to better understanding of the role of P bodies in differential stability of miRNA-targeted mRNAs. As a result, thorough analysis of P body composition would provide more insight into the mechanisms underlying miRNAs function.

EMSY is a recently identified transcriptional regulator that localises to sites of repair following DNA damage and associates with proteins involved in chromatin remodelling. The EMSY gene is amplified in sporadic breast cancer and can silence the transcriptional activation potential of the breast cancer susceptibility gene BRCA2, a tumour suppressor involved in chromatin remodelling and DNA repair. However, the precise mechanism of action of EMSY in transcriptional regulation remains unclear. Preliminary studies show that EMSY binds an internal, alternative promoter within the human tumour suppressor gene p53 and suggest that it may have a role in the control of cellular behaviour through repression of transcription of p53 isoforms. The fact that both EMSY and the various p53 isoforms are differentially expressed between breast tumours and normal breast tissue suggests the existence of a mechanism by which the above process may determine cell fate. The specific aim of this proposal is to decipher the mechanisms through which EMSY regulates transcription. The proposed investigation will employ a series of basic biochemical, molecular and cell biological approaches and use the regulation of transcription of p53 isoforms as the central biological process through which to study the mechanism of interest. The importance of transcriptional regulation by EMSY in the development of breast cancer will be investigated and the roles of BRCA2 and certain chromatin remodelling enzymes in this mechanism directly addressed. This work may define a novel mechanism of transcriptional regulation and provide a deeper understanding of the events involved in the development of breast cancer.

One long standing interest of mine is to understand the mechanisms by which cells interpret and respond to their environment. One such mechanism is direct interaction between neighboring cells. In the context of a developing embryo cell-cell interactions are involved in a variety of processes from tissue morphogenesis to control of organ size and is achieved by a limited number of signaling pathways. The goal of this proposal is to address a fundamental question in developmental biology: How are signaling pathways integrated at the cellular level to give rise to a fully functional organism? Over the last few years, I have been studying Eph receptors and ephrins, evolutionary conserved proteins that provide instructive interactions between neighboring cells. Recently, these studies led me to develop an interest in a different type of cell-cell interaction: direct cell coupling via gap junction communication. Recent studies suggest that Eph receptors and ephrins are potent modulators of stem cell proliferation and apoptosis. The mechanistic details of this regulation remain to be characterized. There is currently tremendous interest in understanding mechanisms regulating maintenance vs. differentiation of NPCs, because of their therapeutic potential. Hence, I chose this system as a model for studying mechanisms of Eph/ephrin signaling in vivo. We will use mouse molecular genetics, cell biology, biochemistry and molecular biology to tackle these questions. The CDA will be invaluable to establish my research group as it will provide both international recognition -which will attract trainees- and financial support to develop my research program.

The complex shaping process of embryogenesis is highly regulated both in time and space at the single-cell level, involving a wide range of control mechanisms. In mammals, most of them are at present unknown or not completely understood due to the lack of reliable in vivo description of cell movements. My project is aimed at imaging whole living mouse embryos in order to map cell movements during gastrulation. I will address the major difficulties that have to be overcome to do this: phototoxicity of imaging, and image quality sufficient for cell segmentation. Phototoxicity will be reduced by performing 2-photon excited fluorescence imaging using an excitation wavelength of 1230nm, which has proven much less destructive for embryos than visible or near-infrared light. I will improve image quality by developing an automated adaptive aberration correction of the excitation wavefront, using an original estimation of wavefront deformation. Finally, I will use these enhanced techniques to study dynamic events during mouse gastrulation, with particular attention to cell movements during the formation of the primitive streak, the three primary germ layers, and elongation of the notochord. This combination of various innovative techniques in optics and embryology should prove powerful to overcome the current limitations of mouse embryo imaging and trigger the development of a complete dynamic atlas of cell fate of highest interest for the comprehension of cell fate regulation in mammals. This interdisciplinary project will provide me new insight both in optics and embryology and will complete my previous research experience in those fields.

Our hypothesis is that the activities of the BDNF TrkB receptor are regulated by recruitment of ubiquitin-interacting proteins that promote internalization, endosomal trafficking and degradation. A recent proteomic approach revealed that among other factors, Hrs and STAM are activated upon BDNF treatment of cortical neurons. Both proteins have established roles in protein sorting and downregulation of the tyrosine kinase receptors EGFR, PDGFR and c-Met, but their importance in neurotrophin receptor trafficking has not been investigated. I will use biochemical and light microscopy techniques to fill this gap and also address at which stage prior to degradation the signal is switched off by monitoring the phosphorylation status of TrkB. In addition, to identify the ligase responsible for TrkB ubiquitination, I will perform an siRNA screen on a human neuroblastoma cell line, using a library that targets ubiquitin ligases. Emerging candidates will be verified by biochemical methods and light microscopy in hippocampal neurons, and their effect on neuronal survival will be tested. BDNF also has an established role in modulating synaptic plasticity. Therefore, in collaboration with Prof. O. Arancio’s laboratory at Columbia University, the relevance of the most promising candidates regulating TrkB/ BDNF degradation will be tested ex vivo by electrophysiological measurements, such as LTP, synaptic fatigue or post-tetanic potentiation, on brain slices. This way we will elucidate if alterations in TrkB and BDNF turnover lead to defects in synaptic transmission. This represents a significant departure from my previous research, which was strictly focussing on cell biology.

IKKalpha, one catalytic subunit of the IkappaB kinase (IKK) complex involved in NF-kappaB activation, is a molecular switch that controls epidermal differentiation. Interestingly, this unexpected function of IKKalpha requires nuclear translocation but not its kinase activity, and is independent from NF-kappaB signaling. IKKalpha–/– mice present a hyperproliferative and undifferentiated epidermis characterized by complete absence of the granular layer and stratum corneum. IKKalpha-deficient keratinocytes do not express terminal differentiation markers and continue to proliferate unless complemented with exogenous IKKalpha. The IKKalpha–/– phenotype is similar to that of IRF6–/– mice, suggesting that IKKalpha and IRF6 regulate common targets in keratinocyte differentiation. Recently, the host laboratory has shown that the Myc family member Mad1, which stimulates cell cycle arrest, is a primary target of IKKalpha in keratinocytes. Upon TGFbeta stimulation, IKKalpha interacts with Smad3 to promote its recruitment to the Mad1 regulatory regions, and to activate Mad1 transcription. To investigate how IKKalpha cooperates with Smad3 to induce Mad1 transcription and whether IRF6 participates in the same process, we will determine if IKKalpha binds directly to a specific DNA sequence in the Mad1 regulatory region and whether it functions to stabilize the formation of a transcriptional complex containing both Smad3 and IRF6.