CELL TO CELL COMMUNICATION AND POPULATION DYNAMICS IN EMBRYONIC NEURAL STEM CELLS – STEMDYN

The mature mammalian cortex is organized in 6 distinct neuronal layers that are composed of different types of projection neurons and interneurons exhibiting specific molecular identities, morphologies and wiring capacities. Cortical projection neurons are produced by sequential waves of differentiation of neural progenitors (NPs) over the course of embryonic development. There is a striking correlation between the date of birth of projection neurons, their identity and their position in the mature cortex, such that first born neurons are positioned internally and late born neurons externally. Thus, development of the neocortex is a complex process that requires coordination between specification and differentiation programs which need to integrate spatial and temporal components.
Integration of these programs within individual NPs and how they are coordinated at cell population level are not fully understood. Specifically, mechanisms by which time is translated into the precise developmental sequence of production of projection neurons in the neocortex are still open questions. Seminal heterochronic transplantations of NPs in the ferret highlighted the important role of external signals in the sequential production of projection neurons and in vitro experiments indicated that temporal information is intrinsic to their lineage. Accordingly, a number of genetic studies indicate that newborn neurons exert a feedback control on NPs decision to differentiate or self-renew, thus regulating the final neuronal output. In addition, our recent work clearly demonstrated that local communication between neighboring NPs via Eph:ephrin signaling also plays a crucial role in regulating timing of NP self-renewal vs. neuronal differentiation. Of specific relevance, we recently obtained preliminary results showing that conditional excision of ephrinB2 in the NP lineage leads to the partial loss of late born neurons but not early born neurons. Our published and preliminary data raise the exciting possibility that temporal variations in Eph:ephrin signaling may constitute an environmental switch controlling the sequential production of projection neurons in the developing neocortex.
The objective of our project is to make a comprehensive decryption of Eph:ephrin signaling in NPs over the course of neocortex development. Our strategy is to characterize the role of Eph:ephrin signaling at the level of cell populations, cell-cell interactions and gene networks.
As a paradigm, we will study the production of the two main projection neuron populations (Tbr1+ early born neurons and Satb2+ late born neurons) from two distinct populations of NPs (Pax6+ apical progenitors and Tbr2+ basal progenitors). We will quantify and model the evolution of these 4 populations in wild type and ephrin mutant embryos at 4 key stages of development. We will perform multiclonal analysis, exploiting the brainbow technology to distinguish cell autonomous vs. non cell autonomous roles of Eph:ephrin signaling in NPs. Further, we will engineer novel tools reporting on the in vivo activity of Eph:ephrin signaling at single cell resolution. Lastly, we will use whole genome analyses to identify transcriptional programs and candidate target genes regulated by Eph:ephrin signaling in NPs. Data collected at single cell resolution and molecular resolution will be progressively integrated into the mathematical model, and the model will be used to choose pertinent time windows for functional exploration of candidate target genes.
As a result of our project, fundamental principles governing the sequential deployment of diverse cellular identities from a population of stem cells will be highlighted; novel molecular tools for Eph:ephrin research will be generated and young scientists with interdisciplinary expertise will be trained.