All the cells of a developing organism carry the same instructions for making proteins, yet the execution of the genetic program is systematically varied to produce the specialized tissues that comprise the fully-formed adult. While the character of each cell reflects the expression of many thousands of genes, this expression pattern is itself determined by a smaller network of regulatory genes, or 'transcription factors.' The causal basis for cellular differentiation can be traced back to the activity of these regulatory networks. For example, in response to diffusive signals called cytokines, self-renewing hematopoietic stem cells give rise to daughter cells that commit to progressively more specialized fates, ultimately becoming terminally differentiated cells, such as erythrocytes, macrophages, and the B and T cells of immunity. This project will develop and apply a new technology - a digital RT-PCR assay using silicone elastomer microfluidic chips - to take a focused look at the behavior of genetic regulatory networks in individual developing blood cells. This research will profile the activity of networks of genes implicated as hematopoietic regulators with single-cell resolution. A database of such profiles will reveal how network activity varies within ostensibly uniform populations of progenitor cells and support comparisons between distinct, developmentally-staged populations. RT-PCR readings will be collated with flow cytometry data to facilitate population analysis that encompasses both surface marker phenotype and internal regulatory network state. Sibling assay studies will be conducted to explore the possibility that metastable regulatory network states bias lineage choice responses to instructive signaling from cytokines.
Broader Impact: This project will further creation of new, interdisciplinary approaches to biological investigations by integrating mainstream developmental genetics, advanced 'lab-on-a-chip' technology, and a quantitative, engineering-oriented approach to the analysis of complex systems. Software developed for the project, including a powerful new program for designing multiplexed PCR primers, will be made available to the research community on an open source basis. In addition, this project will provide opportunity for cross-disciplinary education and training of graduate students; undergraduate students will be involved through a CalTech- sponsored summer research fellowship program (SURF). Valuable research experience will also be available to underrepresented groups through investigator involvement with the Pasadena City College Biotech Program that sponsors research science internships by providing opportunity for interns to work on this cross-disciplinary project.
