Spatial gradients enable cells to precisely organize into complex multicellular patterns. Morphogens are released by clusters of cells and diffuse outward to form a gradient. Surrounding cells convert this gradient into patterns of gene expression using regulatory genetic programs composed of integrated genetic logic, feedback, and cell-cell communication. The central objective of this proposal is to identify the design principles by which genetic programs convert gradients into patterns of gene expression. Our approach will harness two new tools. The first is a set of red and green light sensors that activate a signaling pathway in a graded manner as a function of light intensity. These will be used to deliver light gradients that will be processed as inputs by a genetic program. The second is a suite of computational methods that enable us to convert a desired genetic program into a DNA sequence. This model-guided design allows us to enumerate and evaluate many genetic programs in silico and then construct and experimentally test a library of the most interesting programs. Further, a biophysical method that predicatively tunes expression levels by changing the ribosome binding site sequence will be harnessed to quantitatively compare the mutational robustness and evolvability of programs.
The Aims are organized around the testing of hypotheses as to how regulatory networks process single and multiple gradient signals. Specifically, we will test the following theories: (i) Embedded feedforward loops improve robustness to mutations that affect expression levels (Aim 1). (ii) Feedback loops and spatial bistability increase pattern sharpness and robustness (Aim 1). (iii) Programs that integrate opposing gradients have improved robustness and pattern evolvability (Aim 2). (iv) Opposing gradients can be used as part of size-independent center-finding algorithms (Aim 2). The use of synthetic genetic circuits and E. coli as a model system will make it possible to characterize the gradient-processing properties of many genetic programs and make quantitative comparisons to a fully parameterized mathematical model. This approach will elucidate principles of how regulatory networks are organized to process gradients that can then be generalized to other systems.

Public Health Relevance

The establishment and interpretation of a morphogen gradient is a critical component of many biological phenomena, notably during development, where gradients are central to the establishment of the body plan. Aberrant morphogen release or mutations that affect the regulation controlling their perception lead to human disease, including cancer growth and metastasis, and neuronal and developmental defects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM095765-01
Application #
8022971
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Haynes, Susan R
Project Start
2011-01-01
Project End
2011-07-31
Budget Start
2011-01-01
Budget End
2011-07-31
Support Year
1
Fiscal Year
2011
Total Cost
$146,193
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Mimee, Mark; Tucker, Alex C; Voigt, Christopher A et al. (2015) Programming a Human Commensal Bacterium, Bacteroides thetaiotaomicron, to Sense and Respond to Stimuli in the Murine Gut Microbiota. Cell Syst 1:62-71
Stanton, Brynne C; Siciliano, Velia; Ghodasara, Amar et al. (2014) Systematic transfer of prokaryotic sensors and circuits to mammalian cells. ACS Synth Biol 3:880-91
Nielsen, Alec A K; Voigt, Christopher A (2014) Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol Syst Biol 10:763
Stanton, Brynne C; Nielsen, Alec A K; Tamsir, Alvin et al. (2014) Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat Chem Biol 10:99-105
Segall-Shapiro, Thomas H; Meyer, Adam J; Ellington, Andrew D et al. (2014) A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase. Mol Syst Biol 10:742
Brophy, Jennifer A N; Voigt, Christopher A (2014) Principles of genetic circuit design. Nat Methods 11:508-20
Yang, Lei; Nielsen, Alec A K; Fernandez-Rodriguez, Jesus et al. (2014) Permanent genetic memory with >1-byte capacity. Nat Methods 11:1261-6
Chen, Ying-Ja; Liu, Peng; Nielsen, Alec A K et al. (2013) Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10:659-64
Lou, Chunbo; Stanton, Brynne; Chen, Ying-Ja et al. (2012) Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat Biotechnol 30:1137-42
Moon, Tae Seok; Lou, Chunbo; Tamsir, Alvin et al. (2012) Genetic programs constructed from layered logic gates in single cells. Nature 491:249-53

Showing the most recent 10 out of 15 publications