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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2015)

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).

  • 3 supplemental videos
Cover page: Nodal Gradient Interpretation During Zebrafish Germ Layer Patterning
  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2016)
We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms.
    • Article
    • Peer Reviewed
    UC Merced Previously Published Works (2017)
    Here, we describe an optogenetic gene expression system optimized for use in zebrafish. This system overcomes the limitations of current inducible expression systems by enabling robust spatial and temporal regulation of gene expression in living organisms. Because existing optogenetic systems show toxicity in zebrafish, we re-engineered the blue-light-activated EL222 system for minimal toxicity while exhibiting a large range of induction, fine spatial precision and rapid kinetics. We validate several strategies to spatially restrict illumination and thus gene induction with our new TAEL (TA4-EL222) system. As a functional example, we show that TAEL is able to induce ectopic endodermal cells in the presumptive ectoderm via targeted sox32 induction. We also demonstrate that TAEL can be used to resolve multiple roles of Nodal signaling at different stages of embryonic development. Finally, we show how inducible gene editing can be achieved by combining the TAEL and CRISPR/Cas9 systems. This toolkit should be a broadly useful resource for the fish community.
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