In the developing brain of flies and vertebrates alike, neural stem cells proliferate giving rise to a stem cell population arranged in a columnar epithelium. At some point in time, a wave of differentiation moves across the epithelium, whereby cells leave the epithelial sheet and split asymmetrically into a self-renewing cell and a more differentiated neuronal precursor that will produce neurons. If the transition from proliferative symmetric division to asymmetric division is not triggered at the proper time, the correct number and array of neurons is not achieved. It is therefore essential to understand the underlying molecular mechanisms controlling the proliferative versus differentiated states. Here we focus on the gene network that regulates neural stem cell maintenance in the Drosophila larval brain. Although several signaling pathways are known to be involved, one of which, Notch, is known to be conserved with mammals, there is no global view of the regulatory gene network that maintains stem cell identity, and how this changes upon differentiation. We recently found that the transcription factor Zelda is required for neural stem cell maintenance and inhibits differentiation. We had previously identified Zelda as the "zygotic-genome activator" as its binding precedes and predicts nearly all transcriptional activity in the blastoderm embryo. It organizes sub-networks and establishes many feed forward loops. Zelda also works together with the patterning transcription factors to establish robust and precise expression of their downstream targets, thus playing a comprehensive role in the early gene network. Zelda acts like a pioneer factor, binding target enhancers early and facilitating the binding of other factors that mediate stage and tissue specific activation. Here we hypothesize that Zelda plays a similar role in the neural stem cell gene network. In addition to genetic assays to determine how Zelda interacts with the known signaling pathways, we propose to use genome-wide approaches to identify all Zelda target genes in neural stem cells. This discovery-based approach will yield a comprehensive set of genes with which to build a global gene network. This pilot study is within the scope of the R03 mechanism as it will provide the basis for future studies to further delineate functional connections in the network, first in the Drosophila model system, and then in mammals, since important interactions are likely conserved. The studies outlined in this proposal have the potential to significantly advance our understanding of neural stem cell differentiation, with high impact in the field of developmental neurobiology.
In recent years, stem cells in the developing brain have been identified and studied in model organisms ranging from Drosophila to mouse. Drosophila with its genetic amenability, molecular genetic tools, and the relative ease of collecting and analyzing tissues, has been successfully developed as a model system to study stem cell maintenance and differentiation. We found that maintenance of stem cells is regulated by the transcription factor Zelda, and by identifying downstream targets of Zelda, we will reveal the regulatory gene network underlying stem cell maintenance that are likely conserved with mammals.