Oates+Project

= = = Segmentation and genetic oscillators = = = PI: Andy Oates = = = Biological problem = The segmentation clock is a population of cellular genetic oscillators that generate a collective rhythm used by the vertebrate embryo to segment its body axis. Single cells seem to be capable of oscillation, and a transcription-translation negative feedback loop (pacemaker circuit) involving the Her7 DNA-binding repressor protein is thought responsible. In the tissue, cells are locally synchronized by intercellular communication, a function provided by Delta-Notch signaling. In Her7 mutants, morphological segmentation fails and the segmentation clock becomes spatio-temporally disorganized, but the underlying cause is not understood. Is it because Her7 is required cell autonomously for oscillation, or might Her7 instead primarily control the synchronization of neighboring cells? We will first characterize the dynamics of the segmentation clock’s oscillations in a Her7 mutant at single cell resolution. We will then test the cell-autonomy of the phenotype by following single cells after reciprocal transplantation between mutant and wildtype. We will compare the behavior of transplanted single cells with and without Delta-Notch signaling, to determine whether coupling is required for the observed effects. Together these experiments will be the first single-cell observations of any segmentation clock core-circuit mutant, and will directly test our current models of the cellular and tissue-level organization of the segmentation clock. = Microscopy = - Nikon A1R or Zeiss 700, wavelengths for excitation: 515 562 nm. Filters optimized for YFP and mCherry, a third channel used to distinguish transplanted cells. - 40x, NA= 1 (or better), water immersion objective. - Record timelapse z-stacks of at least 2 embryos at a time, with spatiotemporal resolution (dx,dy < .6 µm, dz < 1.5 µm, dt < 3 min).

= Data Analysis = - Segment growing tissue at single cell resolution and reconstruct track of each cell over time. - Reconstruct temporal evolution of oscillator signal in individual nuclei and compute correlation functions.

= Physical Model = We currently use two physical models of the segmentation clock at different levels of description. To describe the tissue-level organization of the population of oscillators, we use a model of coupled phase oscillators that neglects the microscopic structure of the biochemistry and genetic regulatory steps (Morelli et al., Delayed coupling theory of vertebrate segmentation. //HFSP Journal,// 2009 Feb 3(1):55-66). To describe the core pacemaker circuit of the zebrafish segmentation clock, we use a ODE genetic network model that keeps track of the different DNA-binding repressor proteins and their dimers (Schröter et al., Topology and dynamics of the zebrafish segmentation clock core circuit. //PLoS Biology//. 2012, July 10(7):e1001364). Right now, these two models don’t explicitly link. Were we to discover that the her7 phenotype is primarily a synchronization problem, we would have to consider a direct link between the structure of the core pacemaker circuit and the tissue level synchronization of the clock. = Skills that students acquire =

Zebrafish breeding, embryo staging and mounting, confocal microscopy, image segmentation, cell transplantation. Patience. Persistence. Adjusting to low expectations.