Neural stem cells (NSC) have a fascinating biology of precise transition of cellular states from dormant to proliferative to differentiating, which most recently begun to be understood at the cellular and molecular levels. Despite successful identification of genes linked to each of those cellular steps, a comprehensive and multi-dimensional picture on how those genes are regulated in real-time throughout the differentiation stages of NSC is sorely lacking. This multi- disciplinary proposal will combine for the first time the use of two powerful methods to directly assess the transcriptional state of key genes involved in NSC regulation in vivo, at a single cell resolution. The first technique, Multiplex in situ hybridization, co-developed by one of the PIs, allows direct visualization of transcriptional activity of several genes with single cell resolution. The second technique, known as G-Trace, will be used to generate cellular clones labeled with fluorescent proteins that distinctively mark cell lineages according to their temporal generation.
In Aim 1, the combination of these two methods in the Drosophila larval brain will be used to create profiles of normal wild type expression of several genes involved in NSC regulation simultaneously. Further computational image analysis tools will be developed to segment images and reduce noise. We will test the application of a spectral bar coding system as a proof-of-principle to expand the detection of hundreds of active genes analyzed at a time.
In Aim 2, brain tumors will be induced by knocking-out key transcription factors to track global alterations in gene expression within labeled clones. The expectation is that these combined methods will reveal key properties of the differentiation progression of NSC into several specific neuronal lineages, with far-reaching implications to a better manipulation of stem cells in general and in other organisms, including humans.

Public Health Relevance

Our studies will increase knowledge on stem cell regulation and has the potential to impact the fields of regenerative medicine and stem cell cancer research. A better understanding of global gene regulation at the single-cell level is expected to enhance methods employed in a number of applications, such as in vitro neural stem cell differentiation to repair tissue after damage and development of probes for detecting early stages of cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21EB016535-02
Application #
8548338
Study Section
Special Emphasis Panel (ZRG1-CB-D (51))
Program Officer
Conroy, Richard
Project Start
2012-09-30
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$190,364
Indirect Cost
$69,113
Name
Case Western Reserve University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Ambrosi, Priscilla; Chahda, Juan Sebastian; Koslen, Hannah R et al. (2014) Modeling of the dorsal gradient across species reveals interaction between embryo morphology and Toll signaling pathway during evolution. PLoS Comput Biol 10:e1003807
Schinaman, Joseph Moeller; Giesey, Rachel Lynn; Mizutani, Claudia Mieko et al. (2014) The KRÜPPEL-like transcription factor DATILÓGRAFO is required in specific cholinergic neurons for sexual receptivity in Drosophila females. PLoS Biol 12:e1001964
Chu, Y; Yang, E; Schinaman, J M et al. (2013) Genetic analysis of mate discrimination in Drosophila simulans. Evolution 67:2335-47
Chahda, Juan Sebastian; Sousa-Neves, Rui; Mizutani, Claudia Mieko (2013) Variation in the dorsal gradient distribution is a source for modified scaling of germ layers in Drosophila. Curr Biol 23:710-6