During each neuroblast division, the cell fate determinants Numb, Prospero and Brat segregate into the smaller basal daughter cell where they prevent self-renewal and induce differentiation (Figure 1B). This occurs because the protein kinase aPKC localizes to the opposite apical side and removes the determinants by phosphorylating their membrane localization domains. At the same time, aPKC associates with microtubule binding proteins to ensure that the mitotic spindle is set up in apical-basal orientation.

As a result, only the basal daughter cell inherits the determinants.In the absence of Brat, Numb or Prospero, differentiation is impaired and both daughter cells retain the ability to self-renew. As a consequence, stem cell numbers grow exponentially and the overgrowing stem cells eventually develop into gigantic lethal brain tumors (Figure 1C). Understanding how defects in asymmetric cell division cause the formation of stem-cell-derived tumors is one of the key questions we are currently investigating.

During the last few years, we have performed genome-wide RNAi screens to identify a large number of genes controlling asymmetric cell division and self-renewal in various tissues. For this purpose we use the VDRC RNAi library, a collection of more than twenty thousand transgenic Drosophila RNAi lines that can be induced in a tissue-specific manner. We have used the collection to analyze neural stem and progenitor cells in both the central and peripheral nervous systems of the fly. Our screens have assigned loss-of-function phenotypes to more than 20% of all protein coding Drosophila genes. As each of our phenotypes is precisely quantified, we can use the results for extensive bioinformatics-based analysis. For instance, we can perform hierarchical clustering to group genes by their potential cellular function and integrate our data with pre-existing protein-protein and genetic interaction data to generate functionally annotated networks for specific biological processes (Figure 2).

As a result, we have discovered 23 new global regulators of the Notch signaling pathway. Computer analysis of the resulting interaction network for the Notch pathway has enabled us to identify nuclear pore and nuclear import complexes as well as the COP 9 signalosome as rate-limiting components. For neural stem cells, our analysis has revealed remarkable roles for alternative splicing, chromatin remodeling, and transcriptional elongation in the control of self-renewal. By combining phenotypic data with gene expression analysis, we try to decode the circuits that regulate self-renewal in Drosophila neural stem cells. Our goal is to describe how the circuits are reprogrammed when one of the two daughter cells is driven towards terminal differentiation, and to understand how defects in this reprogramming event lead to the formation of a lethal stem-cell-derived brain tumor.

Can we transfer our results from Drosophila to mammalian and ultimately to human stem cells as well? To analyze the relevance of our data for mammalian biology, we have focused on the mouse brain. The mouse forebrain develops from a limited number of progenitors, which first expand by symmetric division and then switch to an asymmetric division mode in which they generate one progenitor and one or more differentiating neurons. Many of the genes that control neuroblasts act in this system as well.

To characterize the newly identified candidates from our fly screens, we use in utero electroporation (Figure 3). This technology, by which DNA constructs are co-introduced with GFP by high voltage electricity, enables us to perform lineage tracing and study gain or loss of function phenotypes in a single experiment. We have identified phenotypes for several mouse homologs that suggest a remarkable conservation of the molecular machinery governing asymmetric cell division. Ultimately, we hope that these experiments will enhance our understanding as to how vertebrate stem cells control proliferation and differentiation and how these processes are deregulated in tumor development.