S. D. Hatfield, H. R. Shcherbata, K. A. Fischer, K. Nakahara, R. W. Carthew and H. Ruohola-Baker (2005)

Stem cell division is regulated by the microRNA pathway Nature

One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

Yatsenko, A.S., Gray, E.E., Shcherbata, H.R., Patterson, L.B., Sood, V., Baker, D., and Ruohola-Baker, H. (2007)

 A putative SH3-domain binding motif but not the C-terminal Dystrophin WW-domain binding motif is required for Dystroglycan function in cellular polarity in Drosophila, JBC

The conserved Dystroglycan-Dystrophin (Dg-Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of Dg-Dys complex is poorly understood in any cell type. We now find that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline rich conserved C-terminal region can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW-domain binding motif at the very C-terminus of the protein thought to mediate interactions with Dystrophin, suggesting that a second, internal WW-binding motif can also mediate this interaction. We confirm this hypothesis by using a sensitive fluorescence polarization assay to show that both WW-domain binding sites of Dg bind to Dystrophin in humans (Kd = 7.6 & 81muM, respectively) and Drosophila (Kd = 16 & 46mu M, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted SH3- domain binding site abolishes Dg function in cellular polarity. This suggests that an SH3-domain containing protein, which has yet to be identified, functionally interacts with Dg.

 Dystroglycan is required for polarizing the epithelial cells and the oocycte in Drosophila Development

The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.

Deng WM, Althauser C, and Ruohola-Baker, H. 2001

 Notch-Delta signaling induces a transition from mitotic cell cycle to endocycle in Drosophila follicle cells. Development

In many processes, polyploid cells are generated by a variant of the normal cell cycle, called the endocycle, in which cells reduplicate their DNA without dividing. The transition from a normal mitotic cycle to the endocycle is poorly understood. This transition is regulated by the Notch pathway in Drosophila follicle cell epithelium. Loss of Notch in follicle cells, or of the Notch ligand Delta in the underlying germline, blocks the transition of follicle cells from the mitotic cycle to the endocycle. As a result, mitotic cycling continues and the cells overproliferate, becoming smaller than polyploid wild type cells. Regulation must occur at the transcriptional level because Suppressor of Hairless (Su(H)), a downstream transcription factor in the Notch pathway, is required for follicle cells to enter the endocycle. In the stage 12 egg chamber shown here, Su(H)SF8 mutant cells (lacking Green Fluorescent Protein or GFP) are smaller than wild type cells (green because of nuclear GFP). Armadillo staining (red) outlines the cell boundaries while DAPI staining of DNA (blue) labels all cells. The data demonstrate that Suppressor of Hairless (Su(H)) is required for follicle cells to exit the mitotic cycle.