Research

Work in my group has been focused on understanding the origin of polarity and control of cell fate in development. In recent years it has become clear that genes that have been implicated in human disease play important role in these processes (Ruohola et al., 1991; Ruohola-Baker et al., 1993; Larkin et al., 1996, 1999; Morimoto et al., 1996; Tworoger et al., 1998; Deng and Ruohola-baker, 2000; Jordan et al., 2000; Deng et al., 2001; Deng et al., 2003; Althauser et al., 2003). The goals of our research are to improve understanding of the mechanisms underlying these key biological processes and specifically to gain insight into the function of the "disease" genes that have a significant influence on human health. Our current work is focused on the Notch- and the Dystroglycan-pathways.

1) Notch pathway

Most of our studies of the Notch pathway have been aimed at understanding how this pathway acts in patterning and how it interacts with other signaling pathways (Ruohola et al., 1991; Larkin et al., 1996, 1999; Tworoger et al., 1998; Jordan et al., 2000) . More recently we have discovered that Notch also acts in control of cell division ( Deng et al., 2001; Althauser et al., 2003).

• Notch in patterning and signaling interactions

Over ten years ago we observed that gene groups/signaling pathways that were thought to act only in neurogenesis had a much more general role (Ruohola et al., 1991; Ruohola-Baker et al., 1993, 1994). This sparked a large amount of interest in the field, and follicle cell patterning and signaling from follicle cells to establish oocyte polarity became a highly studied subject. It has become clear that the Notch pathway is required multiple times during follicle cell differentiation. The challenge now is to understand how the Notch pathway interacts with the other signaling pathways that also act in epithelial follicle cell differentiation.

We showed that in addition to neurogenesis, Notch pathway is required for a very different process, the follicle epithelial cell patterning that results in stalk formation between developing egg chambers (Ruohola et al., 1991; Larkin et al. 1996, 1999; Tworoger et al., 1998). We further observed a Mirror-Fringe expression border at the site of Notch activity (Jordan et al., 2000). This was interesting because previous studies in vertebrates and in Drosophila had revealed that Fringe mediated activation of the Notch pathway plays a pivotal role in patterning cell layers during organogenesis. In these processes, a homeobox containing transcription factor is responsible for spatially regulating fringe expression and thus directing activation of the Notch pathway along the fringe expression border. Our studies suggest that this may be a general mechanism for patterning epithelial cell layers (Jordan et al., 2000). At three stages in Drosophila oogenesis, mirror and fringe have complementary expression patterns in the follicle cell epithelial layer, and at all three stages loss of mirror enlarges, and ectopic expression of mirror restricts, the fringe expression pattern, with dramatic consequences for follicle cell patterning. These morphological changes are similar to those caused by Notch mutations. These and other results suggest that Mirror and Notch induce secretion of diffusible morphogens. We identified dpp (TGF- b ) as such a molecule in germarium. Recently we have analyzed the Wingless pathways in this process.

To further investigate the interactions between signaling pathways in follicle cell differentiation we are applying microarray technology (Bryant et al.1999). We have developed a data processing pipeline for cDNA micro-array data and have analyzed the differentially expressed genes in different mutant backgrounds (loss-of-function and gain-of-function). Hypomorphic loss-of-function (LOF) mutants as well as Gal4/UAS targeted over-expression of constitutively active (ca) mutant components were used to perturb the EGF receptor pathway. Analysis of the data revealed that activation of the EGF receptor pathway alters the levels of transducers in Notch-, Ecdysone-, Wingless, TGF- b - and JNK- pathways. These pathways, with the exception of Wingless are known to act in late follicle cells. Since the levels of two components of Wingless pathway, Pangolin (transcription factor) and Sugarless (UDP-glucose dehydrogenase) were altered by changes in EGF receptor pathway we are now further analyzing the function of Wingless-pathway in follicle epithelial cells.

Notch and FKBP

Disruption of protein folding in the ER is the basis of many sporadic and inherited diseases. FKBP, which is a protein from the immunophilin group that has an affinity forFK506 drugs and antibiotics, functions as a chaperone and is involved in protein folding in the ER. In our initial cDNA microarray screens for genes differentially expressed in dorsal follicle cells, we identified Drosophila FKBP (Bryant et al., 1999) and subsequently found that overexpression of FKBP produces defects in dorsal appendage patterning. In addition, we have produced loss-of-function FKBP mosaics to determine which pathways are affected when FKBP is defective and found that follicle cell clones in stage 9 egg chambers show Notch sub-cellular mislocalization. This suggests that FKBP might be involved in the transportation of Notch from the ER. We are in the process of analyzing this cell biological function of FKBP in more detail.

• Notch as a tumor suppressor

A long standing question in development is how cells know when to stop dividing. We have now answered this question in the follicle cell epithelium: the underlying germ line tells the epithelial cells to stop. Furthermore, the control of the cell cycle in this system is carried out by the Notch pathway (Deng et al., 2001). These exciting results are described in detail in the following paragraphs. In many developmental processes, polyploid cells are generated by a variation of the normal cell cycle called the endocycle in which cells increase their genomic content without dividing. How the transition from the normal mitotic cycle to endocycle is regulated is poorly understood. We have shown that the transition from mitotic cycle to endocycle in the Drosophila follicle cell epithelium is regulated by the Notch pathway (Deng et al., 2001). Loss of Notch function in follicle cells or its ligand Delta function in the underlying germ line disrupts the normal transition of the follicle cells from mitotic cycle to endocycle, leading to overproliferation of these cells. The regulation is at the transcriptional level, as Su(H), a downstream transcription factor in the pathway, is also required cell autonomously in follicle cells for proper transitioning to the endocycle. One target of Notch and Su(H) is likely to be the G2/M cell cycle regulator String, a phosphatase which activates Cdc2 by dephosphorylation, as String is normally repressed in the follicle cells just prior to the endocycle transition but is expressed when Notch is inactivated. Analysis of the activity of String enhancer elements in follicle cells reveals the presence of an element that promotes expression of String until just prior to the onset of polyploidy, suggesting that it may be the target of the endocycle promoting activity of the Notch pathway. A second element that is insensitive to Notch regulation promotes String expression earlier in follicle cell development, explaining why Notch represses String only at the mitotic cycle-endocycle transition. We are in the process of analyzing the critical string promoter region in more detail.

Because the overexpression of string does not cause extra cell division past the endocycle transition at stage six, there are likely also other components involved in regulating this endocycle switch. To understand how the Notch pathway coordinates this process, we have identified and carried out a functional analysis of genes whose transcription is regulated by the Notch pathway at the transition (Althauser et a., 2003). These genes include the G2/M regulator Cdc25 phosphatase String, a regulator of the APC ubiquitination complex Fzr, and an inhibitor of the CyclinE/CDK complex, Dacapo. Notch represses String and Dacapo, and activates Fzr. The results support a model in which Notch activity executes the mitotic-to-endocycle transition by regulating all three major mitotic checkpoints. Repression of String blocks M-phase, activation of Fzr allows G1 progression and repression of Dacapo assures entry into S-phase. This study provides a comprehensive picture of how external signaling pathways can control cell cycle transitions by the coordinated regulation of cell cycle checkpoints (Althauser et al., 2003).

One of the goals in my group is to reveal whether Notch activity in Drosophila follicle cells impinges directly on transcriptional regulation of string, dacapo and fzr or whether Notch acts through another signaling pathway. Towards this goal and to understand the mitotic-to-endocycle transition better we search for positive regulators of the cell cycle in follicle epithelial cells.

2) Dystroglycan-Dystrophin complex

A gain of function screen in our grouporatory for mutants defective in polarity in Drosophila oogenesis resulted in the finding of three Drosophila homologues of the components in the Dystroglycan complex: Drosophila Dystroglycan (Deng et al., 2003), LamininA (Deng and Ruohola-Baker, 2000) and a novel tetraspanning protein (a possible Sarcospan). Further genome wide sequence analysis revealed that the Drosophila genome has all the known components of the Dystroglycan complex. This complex is part of an evolutionarily conserved machinery found in multiple cell types that allows communication between the extracellular matrix outside the cell and the cytoskeleton inside the cell. In humans, defects in the Dystroglycan pathway causes multiple degenerative muscle dystrophy diseases. However, today very little is known about the regulation of the pathway and no cure exists for the disease.

To analyze the Dystroglycan complex in detail in the Drosophila model system and to understand its function in cellular polarity formation we dissected the function of Dystroglycan. This work (Deng et al., 2003) has led us to propose that Dystroglycan plays a crucial role in the process of establishing polarity and cytoskeletal organization. In particular, defects in the organization of the basal actin are observed suggesting possible connections to the phenotypes observed in muscular dystrophy models. We are taking a multidisciplinary approach towards understanding the function of the Dystroglycan protein in Drosophila. Computational analysis, biochemistry and easy-to-manipulate genetics in Drosophila are used to further dissect the role of the different potential binding partners of the cytoplasmic tail of Dystroglycan in signaling. The long term goal of this project is to better understand the Dystroglycan pathway in hopes that this information can lead to new gene therapy approaches for curing muscular dystrophy.

• Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

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, but how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in Drosophila , we showed that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apical-basal polarity) and the oocyte (anterior-posterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and over-expression 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 set the stage for analyzing the Dystroglycan-complex using the power of Drosophila molecular genetics.

• Dystroglycan in signaling

Recent data has implicated DG in cellular signaling processes, but the regulation and function of the signaling pathways in vivo are not yet understood. The interaction between DG and the cytoplasmic protein Dystrophin is mediated by a PPPY motif in the last C-terminal 18 amino acids of Dystroglycan and a WW domain in Dystrophin. It has been reported in mammalian systems that, when phosphorylated, the Tyr in the PPxY motif blocks DG binding to Dystrophin, while allows the PPxY motif to recruit a SH2 domain containing protein to its cytoplasmic tail. In addition to its structural role, DG has been suggested to mediate cell signaling by interacting with the SH2/3 domain containing protein Grb2. Grb2 is part of the Ras signaling pathway and is involved in embryonic development and malignant transformation; disruption of this signaling pathway can lead to cancer. The Drosophila Grb2 homologous gene is Drk, also an SH2/SH3 adaptor protein. The protein interaction modules of Drk are structurally conserved and functionally interchangeable with those of mammalian Grb2 and the C.elegans Sem-5 genes. Furthermore, a number of SH2 domain containing proteins, most notably Src, interact with the DG C-terminal proline-rich domain and the interactions are dependent on Tyr phosphorylation at the PPPY motif. Taken together, it appears that tyrosine phosphorylation at the PPPY motif regulates selective binding of DG to two different proteins, simultaneously recruiting one molecule and blocking another. The switch of binding between two different proteins indicates that DG may play dual roles in development by using different conduits for downstream pathways.

Taking advantage of this biochemical data in mammalian systems, elegant genetics in Drosophila and modern quantitative analysis methods and biochemistry, we are addressing the following questions in vitro and in vivo in Drosophila. 1) What is the biochemical basis of selective DG binding to Dystrophin, Grb and Src? 2) What is the function of the selective binding? 3) What molecules, other than Dystrophin, Grb and Src interact with DG? Using computer modeling, we have generated the smallest possible changes in Dystroglycan peptide that in vitro still support Dystrophin but not Src binding and are now in process of generating transgenic lines that conditionally express this form of Dystroglycan. The phenotypes of this mutant will reveal the function of selective binding of Dystroglycan to Src. With this same logic we will continue to dissect the biochemistry and function of differential protein binding to the DG C-terminus in vitro and in vivo.

Given DG's implication in such important diseases as DMD, mental retardation and a variety of infectious diseases, any advance in understanding its function in signaling pathway or cellular structure integrity may provide new insight into disease mechanisms and aid new therapy development.

3) Differentiation of the germ line

One of the goals in my group is to take a multidisciplinary approach towards understanding the function of the external and internal stimuli that regulate the differentiation of germ line and in particular, oocytes using Drosophila melanogaster as a model system. During oocyte differentiation, a long arrest in meiosis I is observed from human to mice to flies, however, the molecular basis for this regulated arrest and its release are not fully understood. Since around 80% of human disease genes have homologues in Drosophila, genetic, biochemical and microarray-based analysis of the oocyte differentiation steps in Drosophila should shed light also to the regulation of these birth defect/cancer-critical processes in Man.

As described above, our studies of the Notch and Dystroglycan pathways are shedding considerable light on the differentiation of the germ line in Drosophila. We have also gained insight from studies of a mutant called Maelstrom which was identified through its effect on oocyte polarity. Characterization of Maelstrom showed that it is associated with perinuclear, electron-dense granules called nuage that are a hallmark of germline cells across the animal kingdom. Further genetic and cell biological analysis of Maelstrom suggested a relationship between nuage and the microRNA-pathway and a potential function of microRNAs in translational control in oocytes (Clegg et al., 1997; Clegg et al., 2001; Findley et la., 2003).