Enhanced Transgenics: Bioinformatically Informed Design of Transgenic Tools for Systems Neuroscience

If the brain were so simple we could understand it, we would be so simple we couldn't. -Lyall Watson The mammalian brain is arguably the most complex structure known, with trillions of electrically-active cells interconnected in astonishingly complex ways. Understanding how neural circuits underlie behavior requires having access to their individual components, i.e. neuronal cell types. However, our current understanding of brain function is more at the level of brain regions rather than the neuronal cell types which are their functional constituents. It is not even known how many different kinds of neurons there are in the brain, estimates range from hundreds to hundreds of thousands. Distinct cell types throughout brain and body express differing amounts of different genes depending upon differential activation of promoters, the control regions of genes contained in non-coding sequences. We have developed an approach to dealing with this neural diversity by using components of the genetic machinery that underlies the diversity of neuronal cell types (i.e. enhancers) to create synthetic promoters specific to particular neuronal cell types of targeted brain regions, which we call Enhancer-Driven Gene Expression, or EDGE. This allows us to create molecular genetic tools capable of targeting transgenes to specific neuronal cell types, allowing us to address neural circuits at the level of granularity at which they actually function. When distributed throughout the scientific community, these neuron-specific tools should lead to fundamental advances in the understanding of both normal and pathological brain function, and potentially provide entirely new avenues of treatment of disorders of the central nervous system. In this final award period, we have started to finally use our tools for the purposes they were designed for: performing celltype-specific investigations of the circuitry of memory. These include both anatomical investigations of particular cell types in Entorhinal Cortex and the Claustrum, and finally, manipulating the activity of particular neurons in these brain regions to determine the response in other connected circuit elements. These ongoing studies have led to unique insight into the functional circuitry of memory, the core interest of our group. On the molecular side of the lab, we greatly improved our original approach technically, putting it into the context of modern genomic techniques. We have updated the EDGE approach to incorporate single-cell techniques, most notably scATACseq. Assigning a putative enhancer to a scATAC cluster allows one to choose enhancers enriched in particular cell types, a significant advance from our initial approach. Finally, we have switched our enhancer screen entirely to AAV vectors rather than transgenics, and have already obtained some interesting candidates for both the subiculum and the prefrontal cortex in this way. Not only does this greatly increase the throughput of the EDGE screen (6 months to make a mouse, but just 6 days to make a virus), it also opens up the possibility for translational applications in time.

We have completed all of the primary objectives (1-3), and most of the secondary objectives (4,5) in our proposal. In addition, we also went in an exciting new direction, expanding our technique to AAV vectors, see the Results Report for details. Objective 1, Bioinformatics: We have performed ChIPseq of tissue lysates from 22 different regions of interest in the mouse brain, providing the basis for our enhancer screening. Objective 2, Transgenics: We injected 27 distinct tTA expression constructs containing region-specific enhancers with minimal promoters into oocytes for transgenesis. Objective 3, Anatomy: We anatomically and histologically characterized the expression patterns of each of the 7 lines deemed specific enough in the above screen. Objective 4, Behavior: We have performed a preliminary behavioral test on silencing the lines targeting RSCs. Objective 5, Physiology: We have completed two sets of experiments examining the effects of manipulation of RSC activity.

The mammalian brain is the most anatomically complex structure known, with far more cell types than the rest of the body combined. Indeed, it is not known how many different kinds of neurons exist, or even how to define them- by genes, morphology, connectivity, or receptive field. Understanding this incredible cytological diversity within the brain is central to understanding function. While electrophysiological and lesion studies can illuminate what a particular brain region does, they shed less light upon how it does so. Understanding brain function at this level requires access to the relevant circuit elements i.e. the various cell types comprising that brain region. While arguably transgenesis is the only approach that can operate at this level of anatomical granularity, traditional techniques have led to relatively few successes. We present a multidisciplinary approach called Enhanced Transgenics that allows us to address this fundamental problem: we design transgenic mice based upon enhancers rather than promoters. Modern functional genomic techniques such as ChIP-Seq and RNA-Seq allow identification of gene regulatory sequences in an unbiased, high-throughput fashion. When we applied these technologies to replicates of microdissected brain tissue from a handful of regions of adult mouse brain we discovered a surprisingly large diversity of putative enhancer sequences. Comparing the results from lysates of different brain regions enables identification of RSPEs. The most promising RSPEs are cloned into a minimal-promoter injection construct for transgenesis. Founders that appear to drive expression in distinct RSCs are selected for further characterization. Particularly interesting lines are then mated to effector lines for further anatomical, behavioral and electrophysiological experiments. We concentrate first upon the Medial Entorhinal Cortex, but our approach is transferable to any brain region, truly promising a means to 'take the brain apart'