Nodal Gradient Interpretation During Zebrafish Germ Layer Patterning
This thesis presents work toward understanding how cells within an embryo receive fate and positional information. It concentrates on the Nodal morphogen gradient, which specifies germ layer identity through differing levels of Nodal signaling. Although much progress has been made in understanding the components and outcomes of the Nodal signaling pathway, there is very poor quantitative understanding of how cells translate the duration and concentration of Nodal signaling into fate/positional information. To address this question, several new technologies were employed or developed.
Light sheet fluorescence microscopy was used to image Nodal signaling dynamics in vivo (Chapter 5). This new imaging technology allowed us to: determine which cells are exposed to the Nodal signal, quantitate the duration and level of this signaling, and directly correlate this with cellular response, i.e. which Nodal target genes are turned on and what germ layer fate is adopted. We will use this information to propose an initial model for how Nodal signaling is interpreted at a cellular level. We will test our model with additional data taken in the presence of an inhibitor, to access whether the system behaves as expected when the input is modified.
In order to develop finer control of the spatial and temporal aspects of perturbing a biological system, we simultaneously developed optogenetic tools for use in zebrafish embryos. Most optogenetic systems, especially those that control protein localization and expression, have been developed at the tissue-culture level and do not transfer directly to the multicellular organism level. We were successfully able to optimize two optogenetic systems for use in zebrafish, the phytochrome system for protein localization control (Chapter 2) and the LOV system for transcriptional control (Chapters 3 and 4).