Genetics and genomics of neural wiring
Marta MoreyThe assembly of a functional neural circuit requires that neurons in the network wire correctly. A key question in this process is how neurites discriminate between one another and form connections with their specific synaptic partners. Virtually nothing is known about the genetic and molecular mechanisms that regulate wiring specificity. Expanding our knowledge in this field can help us understand the ontogeny of neurological disorders caused by abnormal circuit assembly, and provide an insight into general principles helpful to coax stem cell derived neurons to properly integrate and reconstruct damaged neural circuits.
To address wiring specificity we use the fly visual system as a model, and take advantage of its striking similarities to vertebrate neuropils, both in terms of neuronal diversity, layered structure and glial support. We combine state of the art genetic techniques, genomic approaches and imaging to unravel how neurons establish specific connections through the following projects.
Neuronal phenotypes are determined by molecules that regulate the morphological, biochemical, and physiological properties of a cell. Thus, the unique phenotype of different types of neurons is expected to be the result of their differential gene expression. Hence, comparison of gene expression profiles between neurons should identify key molecular components that specify their distinct functions. Our laboratory has developed genetic approaches to isolate cell type specific neuronal populations in the fly visual system in a highly purified manner using FACS sorting. Subsequent high through put RNA sequencing allows us to get a grasp on gene expression differences in developmental time courses as well as between different types of neurons. We apply these techniques to answer different biological questions.
Our hypothesis is that the molecular differences between neuronal subtypes, with similar developmental origin and function, contribute to their distinct connectivity. We study well-defined photoreceptor subtypes and lamina monopolar neurons, with different layer and synaptic specificities. Transcriptional profiling has allowed us to characterize the unique combination of mRNAs encoding cell surface molecules expressed in R7 and R8 photoreceptors, and L1-L5 lamina neurons, as well as to identify molecular differences between neuronal subtypes. We use diverse genetic approaches to test the involvement of differentially expressed candidate genes in wiring specificity. For example, through genomic protein tagging we have confirmed that the Dpr family of Ig-domain containing proteins is expressed in unique combinations in homologous neurons with different layer specific synaptic connections. Using genomic protein tagging combined with colocalization of cell type specific markers, we found that a subset of lamina neuron subtype specific synaptic partners express Dpr Interacting Protein family members known to be binding partners of the Dprs expressed in lamina neurons. This exquisite Dpr-DIP expression between synaptic partners suggests that these molecules contribute to their wiring. As part of another project, we are currently performing a large-scale RNAi screen on R8 and R7 differentially expressed genes to identify cell-subtype specific gene batteries determining R8 and R7 wiring specificity. To confirm RNAi results we use clonal analysis on available genetic mutants or CRISPR engineered ones generated in our laboratory.
Our long-term goal is to elucidate the transcriptional logic and architecture of synaptic layer selection genetic programs. Our work identified R7 and R8 cell type specific transcription factors involved in their differential wiring specificity. There is data suggesting that these transcription factors might be working in parallel with other transcription factors expressed in both cell types. We plan to combine our RNAi screen data, i.e. gene batteries involved in layer selection, with in silico studies of their cis-regulatory regions, as well as biochemical studies reflecting differences in chromatin structure. We expect that this three-pronged approach will delineate for the first time molecular and regulatory features of a layer selection program.
The role of glia in regulating neuronal survival had been long recognized. However, this trophic support function has hampered attempts to address additional, more active functions of these cells in the nervous system. There is evidence supporting a role for glia in synaptic development and activity, as well as experiments suggesting a role of glia in guidance of axons and process outgrowth. However, the molecular mechanisms regulating these functions are not fully understood. The development of the fly visual system is intimately related to glia. A particular glial subpopulation, required for the guidance of photoreceptors in the medulla, is the focus of our study. We are investigating the relationship of chloride channels and the signaling physiology of this glia. The knowledge we gain from the fly is expected to help us understand the physiological role of these vertebrate chloride channels expressed in certain glial populations during development and in reactive glia upon injury.
