Ani Arun and Shirley Galbiati 20.109 Proposal

Scientific Questions and Engineering Goals
Inspired by the Hasty lab synchronized oscillator, we would like to construct a synchronized oscillator in mammalian cells. We would like to use this synchronized oscillator to form static spatial patterns of gene expression, as occurs naturally in the vertebrate segmentation process. Our project could shed light on the key processes that govern the vertebrate segmentation in development, as well as serve as a starting point for programmed patterned tissue formation and other applications in regenerative medicine.

In our implementation of the system, we would rely on fluorescent proteins as the readout of cell state. Experimental verification of our system would require time-lapse quantitative imaging techniques.

Background Information
This goal may require the use of a synthetic mammalian cell-cell communication mechanism. Thus far, none has been published. One idea is to use Delta and Notch signaling in cells that have the endogenous copies knocked out or knocked down. (More literature combing on this topic is needed). We would also need to come up with network topologies and a corresponding molecular implementation that could result in robust, synchronized oscillations. Finally, we hope that inspection of the current understanding of the role of FGF8 (fibroblast growth factor 8) in translating temporal segmentation clock pulses to spatial arrangement of segmentation boundaries will enable us to reach our pattern formation goal.

Update 5/3: Components needed for sequential segment patterning:


 * Moving morphogen gradient
 * Locally synchronized oscillation
 * Bistable switch (default state: high morphogen, "on" state (less sensitive to oscillation than morphogen). can be switched "off" at peak of oscillation and low morphogen.

Cool video: http://www.youtube.com/watch?v=p9Ogg8BJW8c&feature=related

Questions:


 * How to implement moving morphogen gradient?
 * How to implement synchronized oscillator?
 * Need mammalian cell-cell communication with minimal cross-talk
 * Need a way to regulate cell-cell communication
 * Need robust topology
 * How to couple synchronized oscillator to bistable switch in the simplest, most robust manner?

Potentially useful model systems
Delta-Notch

Neuronal Signaling- ability to regulate degradation of neurotransmitter

Hormones

Cancer adhesion

Axonal growth- is this genetically regulated?

Other paracrine signalling systems....

Challenges
Balance of parameters to get oscillation- sensitivity of genetic parameters. Similar to other problems in oscillating systems.

Mechanical construction of the system- Hasty relied on fast reproduction rate of bacteria- not an option in mammalian cells.

Thyroxine Model: Update 5.4.11
Thyroxine (T4) is natively synthesized by the thyroid gland and is secreted into blood plasma. When it enters target cells, it controls gene expression by binding to transcription factors within the nuclei. Thyroxin has been found to be involved in controlling the growth and differentiation of cells.

Fire and degrade mechanism: Static biofilm with cells genetically modified to contain a plasmid with all necessary genetic components.

Promoter for fluorescent protein (GFP) is that which is recognized by Thyroxine. Therefore, in the presence of thyroxine, cells will fluoresce. Thyroxine also induces the production of further thyroxine. The exact mechanisms of synthesis will be explored later due to the complicated interplay between thyroglobin and thyroxine.

Need to figure out degradation/inhibition pathway to temporarily inhibit the effects of thyroxine. Degradation system should be intracellular, exceed the half life of thyroxine.

If we provide a mechanical construct to route thyroxine produced by the terminal band back to the start, can form an infinite loop of longitudinally traveling fluorescence.

Presentation Outline: Update 5.4.11
Introduction

Research goal/proposal -	What are we trying to accomplish?

What are some of the underlying principles of mammalian cell-cell communication?

What are the challenges faced in this problem?

What has already been done (cite 2-3 previous studies in depth)? -	How have those researchers addressed the previously stated challenges?

Model 1 -	Explain how it works, why its cool

Model 2 -	Explain how it works, why its cool

Tools/materials needed to develop the system/troubleshooting.

Future applications of the study.