In vertebrate and invertebrate brains, neurons establish regionally distinct circuit architectures. Astrocytes are crucial elements in this environment, displaying remarkably elaborate, yet specific branching patterns. However, how astrocytes acquire their diverse morphologies in interaction with neurons and how these shape neuronal morphologies and function remains poorly understood. Our study will use the Drosophila visual system as an in vivo model, focusing on the interactions of neurons and astrocyte-like glia in the medulla. The latter establish stereotypic columnar and layer-specific branching patterns during development. Previously, we have shown that the glial-specific transmembrane Leucine-rich repeat protein Lapsyn is required for astrocyte branch extension into the synaptic neuropil. Using label-free mass spectrometry, we isolated a member of the Innexin family as one potential binding partner of Lapsyn. Based on our genetic analysis, we hypothesize that neuron subtypes engage in a dialog with astrocytes through gap junctions, that are stabilized by Lapsyn, to establish correct local glial morphologies. These in turn could refine neuronal development and function. In this integrative project from development to function, we will employ tailored genetic tools to achieve cell-type specific labeling and gene manipulations, as well as high-resolution and functional imaging. Specifically, our aims are (1) to characterize the developing neuron-astrocyte interface in the context of gap junction communication with specific neuron subtypes, (2) to assess the dynamic behavior of astrocyte branches during development, and (3) to examine how astrocyte branches shape synaptic connectivity and functional neuronal properties such as neurotransmitter recycling and axon initial segment proteins.

The mammalian neocortex is divided into distinct cytoarchitectonic areas which form functionally specialized neural circuits that subserve important motor, sensory and cognitive functions. A fundamental question is how diverse cortical areas are generated during development. The prevailing model is that these areas arise from an initially homogenous and multipotent neuroepithelium and that signaling centers (the cortical hem (CH), anterior commissure (ACo)/Septum (S) and pallial subpallial boundary (PSB)) secrete instructive cues, or morphogens, which diffuse across the germinal epithelium to provide progenitors with positional information. This regulates the graded expression of transcription factors (TFs) which is crucial for the early regionalization of the cortex and the positioning of boundaries that separate the forming compartments. However the cellular and molecular mechanisms involved in this process are poorly understood. In this project we want to explore the role of homeoprotein (HP) non-cell autonomous signaling and of Cajal-Retzius (CR) cells originating from the CH, S and PBS in cerebral cortex arealization. The project is divided in three primary tasks. Task 1 will explore the paracrine functions of Pax6 in cortical patterning and arealization. The main tool will consist in minigenes encoding secreted single chain antibodies against Pax6 (SaP6). In a first series of experiments the antibodies and control antibodies will be expressed through electroporation and the consequences on arealization will be evaluated by several histological techniques (ISH for layer and area specific genes, corticothalamic and thalamocortical projection tracing). Another approach will consist in crossing mouse lines expressing the CRE recombinase under the control of specific promoters with mice bearing a transgene encoding a Flox-Stop-Flox SaP6s only expressed upon recombination. Analysis of the phenotypes will be as mentioned above. Task 2 will investigate whether secretion is involved in CR cell migration and, consequently, the regulation of patterning. This will be achieved thanks to an original flattened cortex culture model that allows one to follow the migration of genetically labeled CR cells originating from the 3 regions: CH, S and PBS. Secretion will be blocked by pharmacological (toxins) and genetic tools and the consequences of such treatments on CR cell migration and cerebral cortex patterning (ISH for distinct genes, including Emx2 and Pax6) will be studied. The aim of Task 3 is to analyze the reciprocal signaling between CR cells and progenitors with a strong emphasis on Pax6 transfer and its putative implication on the migration of CR cell sub-populations (CH, S, PBS) and the ensuing consequences on graded gene expression (as above). This task also includes an analysis of the molecular mechanisms and, in particular, by a search for a cooperation between CXCL12/CXCR4 and Pax6 signaling. Given the data on Engrailed HP non-cell autonomous activity published by Partner 1 demonstrating that Engrailed signaling involves the local regulation of protein translation, a possible implication of translation regulation by Pax6 and the identification of the mRNAs that enter translation upon Pax6 internalization will be achieved. All in all, this project, if funded, will shed light on the mechanisms at stake in the very important phenomenon of cerebral cortex compartmentalization and function.