In collaboration with pioneer imaging tool builders on campus, we use both primary cell cultures and early developing mouse embryos as complementary and parallel model systems to 1) discover novel gene networks that control cell-fate choices and 2) dissect the molecular and cellular dynamics of gene control mechanisms underlying complex developmental pathways.
Gene Regulation and Genome Organization in single live cells
Critical lineage commitment events during animal development must be exquisitely timed and orchestrated by dynamic molecular transactions of multiple transcription factors in the nucleus. In the past several years, by working closely with Transcription Imaging Consortium (TIC, consisting of the labs of Carl Wu, Robert Singer, and Robert Tjian) and labs of Eric Betzig and Luke Lavis, we first developed a imaging method capable of discriminating specific DNA binding events at endogenous diploid allele sites from non-specific macro-molecular interactions in single-living embryonic stem cells; our single-molecule measurements also describe several key kinetic features (3D diffusion periods, number of trials, search times, residence times etc.) associated with in vivo TF dynamics. Recently, we developed a lattice light-sheet microscopy based single-molecule imaging strategy to map and functionally probe TF binding site spatial organization in single, living stem cells. In the next step, we plan to team up with multiple external TIC labs in a highly collaborative fashion to optimize new molecular imaging systems to study mechanisms linking TF dynamics, genome organization and transcription kinetics. Furthermore, with the help from labs specialized in imaging instrumentation on campus, we would like to devise fast multicolor imaging strategies to study the 4D Nucleome (architecture and dynamics) in single live cells.
Linking Gene Regulation and Developmental Dynamics
A hallmark of nervous system development across bilateral animals is the internalization of neural precursor cells to form ventral or dorsal nerve cord along the anterior-posterior (AP) axis. Specifically, in mammals, the initial formation of neural tube requires extensive interplay between neuro-ectoderm and a transient structure of the axial mesoderm – the notochord. Currently, the transcription circuitry that drives the coordinated cellular dynamics and lineage diversification during neural tube and notochord development is still largely unknown. As a long-term project aimed at quantitatively elucidating gene regulation mechanisms underlying mammalian nerve system/notochord development, we have systematically characterized the developmental pathway mediated by a key developmental regulator – Brachyury during early mouse embryogenesis. Brachyury is a classic enhancer binding TF critical for mesoderm lineage specification and notochord formation. In the next step, we would like to work with labs of Philipp Keller and Tzumin Lee and use a novel combination of cutting-edge developmental imaging and single-cell genomics to characterize the dynamic interplays between neural tube induction and notochord development. Eventually, by correlating the imaging dataset to systematic genome-wide datasets, we hope to learn new mechanistic insights into the molecular signatures associated with certain types of cellular behaviors identified in the whole-embryo imaging experiments.
Interfacing with Neuroscience
Gene regulation defects underlie neural disorders such as Huntington’s disease and Rett syndrome that deteriorate brain structures and their functions. However, it remains largely unknown how genetic mutations eventually alter molecular dynamics to influence gene regulation in distinct neuron populations and what are the downstream effectors responsible for specific disease phenotypes. We are currently working with both on-site and off-site collaborators to characterize molecular dynamics associated with specific genetic mutations. We plan to establish a robust microscopy platform to study single-molecule dynamics in neurons from both primary cultures and acute brain slices.
Finally, we envision heterogeneous molecular distribution and dynamics also actively contribute to large-scale neural computation by influencing the basic biophysical properties of single neuron units in the network. We are looking forward to working with our colleagues at Janelia to test this hypothesis.