Molecular “flight data recorder” / molecular memory device (write-read technology)
Seth Shipman is collaboratively developing (with George Church, Jeff Nivala) a DNA-based equivalent of a “flight data recorder”– a “molecular memory device” for each differentiating cell, to identify both multi-stage probabilistic and/or rare stochastic events, and to record these into the DNA and read back the transcriptional history of development through sequential checkpoints of appropriately successful neurons, and of cells that make incorrect “choices” and/or that get “confused”, chimeric, hybrid and don’t mature correctly. To elucidate and then potentially control development and maturation of neurons, Seth is developing this capability to record ongoing transcriptional activity as cells/neurons pass through maturational “checkpoints” and phases (appropriately or inappropriately), commit to fates (defined or “confused”), and mature. Ideally, this technology will resemble a “flight data recorder”, enabling molecular interrogation of individual cells’ histories and maturation– not at single, static times as is standard, but throughout maturation– as they progress from pluripotency in culture or progenitor stages in vivo to specific, differentiated neuronal identity, or stalling, or confusion.
- Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547, 345–349 (2017). Pubmed
- Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. Molecular recordings by directed CRISPR spacer acquisition. Science 353, aaf1175 (2016). Pubmed
Molecular timer technology
We are developing approaches to convert asynchronous development into linear maturational time via encoded molecular clocks. It is important to know the sequence and timing of developmental stage advancement and transcriptional networks, but that is currently obscured by pooled analyses; there is asynchrony of maturation, both in vivo and of cells/neurons in culture, e.g. those derived from iPSCs/ESCs. These approaches and technology will enable synchronization of developmental and maturational analysis via protein timers linked to sequential developmental events, in vivo or in culture. To understand (and potentially control) with optimal precision the transitional moments in acquisition of cell fate and neuronal development and maturation, we should optimally identify and group same-stage cells, at particular maturational stages, for analysis. Understanding transcriptional and other networks that control cell fate decisions endogenously will likely be enabling in driving differentiation of progenitors and stem cells into mature, subtype-defined neurons.
Molecular technology to interrogate transient cellular interactions during axonal pathfinding; “guidepost” interactions during circuit development
We are developing approaches aimed at discovering neurons’ interaction partners during axonal extension and neuronal maturation, likely reciprocally regulating connectivity and circuit formation. Neurons respond both to peptides and activity at their cell bodies, and in developmentally stage-specific ways to parallel regulators where axons (and dendrites) interact with cells along the way. These interactions are likely critical in advancing through sequential stages of development and circuit formation, but remarkably little is known about these processes in vertebrates. Further, there are likely interactions about which we want to know more than “where and when”, but “what and how” interacting cells signal during normal development– and to identify mechanisms for in vivo and in vitro directed development of subtype-specific neurons and their circuitry.
- Poulopoulos, in preparation
New technologies and tools for genetic mosaic functional analysis:
To enable powerful and currently unavailable or limited genetic mosaic investigation of gene function in complex cellular systems, in particular development and diversity of neuronal subtypes and their complex circuitry in cerebral cortex, we have developed and are further developing and implementing two entirely novel, inter-related systems for genetic mosaic functional analysis. These systems enable binary– all-or-none, neuron-by-neuron “aleatory”– random– mosaic analysis with control over ratios of wt and genetically manipulated cells. These new systems will substantially extend the range of tools available for mosaic analysis. The first is a plasmid-based transfection system, BEAM (for binary expression aleatory mosaic), which generates two genetically distinct, non-overlapping populations of cells for comparative analysis. We are further developing and adapting BEAM for viral use. BEAM can be used directly on wild-type or floxed mice, without the need for complex breeding schemes. We are also a developing a related system of engineered BEACON mice (for binary expression aleatory cre-operated nested mosaic), generating distinct populations of green cells and red cells, with parallel gene modification. BEACON mice can be used to delete genes of interest and/or to activate expression of effector molecules using existing alleles, either throughout the entire organism, or in a specific organ or cell type of interest. The motivating biological goals of this technology development are both to elucidate central molecular controls and regulatory mechanisms over development, subtype diversity, circuit formation, and potential regeneration of neocortical projetion neurons, and to identify potential causes and therapeutic approaches to their dysgenesis and disease.
- Greig, in preparation