I) miRNA function in Stem Cells
One of the key characteristics of stem cells is their capacity to self-renew throughout the lifetime of an animal. Stem Cell self-renewing division is tightly controlled process; too little division disrupts the homeostasis of the tissue while too much can result in cancer. We and others have recently shown that miRNAs are required for germ line stem cell division in Drosophila (Hatfield et al., 2005; Shcherbata et al ., 2006). We are now in the process of identifying and analyzing the regulation of critical miRNAs in stem cell division
1) miRNA function in Human Embryonic Stem Cells (HESCs)
We are in the process of characterizing the microRNA expression profiles and testing the function of the key miRNAs in Human Embryonic Stem Cells (HESCs).
microRNAs are small (20-23nt) RNA molecules that can regulate gene expression. Previous work has demonstrated that miRNAs are required for normal stem cell function in mouse, Drosophila and plants. In addition, miRNA function is implicated in cancers. These two themes might connect as cancer is thought to derive from cancer stem cells. We now proceed toward understanding the function of microRNAs in HESCs in hopes of better controlling HESCs in future applications, including regenerative medicine
miRNAs, small non-coding RNA molecules are transcribed by Pol II from endogenous genes as pri-miRNAs that contain 5’ 7-methylguanosine cap and 3’ polyA-tail. These pri-miRNAs are processed by Drosha in the nucleus to produce a stem-loop-containing pre-miRNA. The pre-miRNA is then transported out of the nucleus, further processed by Dicer to mature 223nt long miRNAs, and incorporated into the RNA induced silencing complex (RISC). The mature miRNA in RISC then provides the information that results in silencing of target mRNAs. To date, 462 human miRNAs have been isolated (http://microrna.sanger.ac.uk/sequences/index.shtml ), however, the cellular processes in which each of these miRNAs participate are poorly understood. We have shown that miRNAs are essential for stem cell self-renewal in Drosophila (Hatfield et al., 2005). Our goal is now to identity of the critical miRNAs for stem cell function in HESCs.
The goal of the project is to generate a miRNA fingerprint for different HESC lines and to identify candidate miRNAs for further functional analysis in HESCs.
2) miRNA function in Drosophila Germ line Stem Cells (GSCs)
We are in the process of characterizing the microRNA expression profiles and test the function of the key miRNAs in Drosophila Germ line Stem Cells (GSCs).
To date, 78 Drosophila miRNAs have been isolated (http://microrna.sanger.ac.uk/sequences/index.shtml ), however, the cellular processes in which each of these miRNAs participate are poorly understood. We have shown that miRNAs are essential for stem cell self-renewal in Drosophila (Hatfield et al., 2005). Our goal is to identity of the critical miRNAs for this process in Drosophila as well as in HESCs.
The first goal of the project is to analyze miRNA expression in Drosophila Stem Cells using sensor-constructs and LNA in situs to identify the candidate miRNAs. The second goal is to test the in vivo function of these candidate miRNAs in GSCs taking advantage of the easy to manipulate genetics of Drosophila.
II) Drosophila as a model for Human Diseases
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; Jordan et al ., 2005;
Althauser et al., 2005; Ward et al., 2006). More recently we have
discovered that Notch also acts in control of cell division (Deng et
al., 2001; Schaeffer et al., 2004; Shcherbata et al., 2004; Jordan et
al., 2006).
1.1. 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 (Jordan et al., 2005; Ward et al., 2006).
To further investigate the interactions between signaling pathways in follicle cell differentiation we are applying microarray technology (Bryant et al.1999; Jordan et al., 2005). 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.
1.2. 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; Schaeffer et al., 2004;
Shcherbata et al., 2004; Jordan et al, 2006). 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.
2) Dystroglycan-Dystrophin complex
Dystroglycan
(DG) is a key element of the dystrophin associated glycoprotein complex
(DGC), which is closely linked to pathogenesis of several forms of
muscular dystrophy. However, the basic cellular function of DG complex
is largely unknown. The overall objective of this project is to study
the protein-protein interactions in which DG is involved in and their
implications for cellular signaling. The extracellular matrix and
cytoskeletal network are intricately interconnected, providing the cell
with both structural integrity and a means for signal transduction. As a
transmembrane protein, DG provides a physical link between the extra
cellular matrix and cytoskeleton by attaching to laminin-2 at its
N-terminus and to the cytoskeletal protein Dystrophin at its C-terminus.
Recent evidence has implicated DG in cellular signaling processes by
binding to SH2/SH3 domain containing proteins, but the downstream
signaling pathways are not clearly understood. We have dissected the
muscle and neuronal disorders in Drosophila muscular dystrophy and
analyzed the binding motifs in DG that are required for function (Deng
et al., 2003; Shcherbata et al., 2006; Gray et al., 2006). We are now
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.
2.1. 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) (Deng et al., 2003). 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.
2.2. Muscular Dystrophy in Drosophila
We have now shown that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex (Shcherbata et al., 2006). Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, our work has now revealed that Dg interacts with Insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is an important identification of a signaling pathway interacting genetically with Dg.
2.3. Dystroglycan in signaling
We
find using overexpression and loss-of-function rescue-assays in
Drosophila that more than half (34 residues) of the Dg proline rich
conserved C-terminal region can be truncated without significantly
compromising its function in cellular polarity. 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 showing with a sensitive fluorescence
polarization assay that both WW-domain binding sites of Dg bind to
Dystrophin in humans (Kd = 7.6 & 81mM, respectively) and Drosophila
(Kd = 16 & 79mM, respectively). In contrast to the insensitivity to the
large deletion, one mutation to an alanine of a proline within a
predicted SH3 domain binding site abolishes Dg function, suggesting that
as yet unknown SH3 domain containing protein interact functionally with
Dg (Gray et al., 2006).
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 laboratory 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).
Selected Publications
Shcherbata, H.R., Hatfield, S., Ward, E.J., Reynolds, S., and Ruohola-Baker, H. (2006) The MicroRNA pathway plays a regulatory role in stem cell division. Cell Cycle 5:172-5.
Jordan, K.C., Schaeffer, V., Fischer, K.A., Gray, E.E., and Ruohola-Baker, H. (2006) Notch signaling through Tramtrack bypasses the mitosis promoting activity of the JNK pathway in the mitotic-to-endocycle transition of Drosophila follicle cells, BMC Dev. Biol. 6:16
Ward, E.J., Zhou, X., Riddiford, L.M., Berg, C.A., Ruohola-Baker, H. (2006) Border of Notch activity establishes a boundary between the two dorsal appendage tube cell types. Dev.Biol. [Epub ahead of print]
Shcherbata, H.R., Yatsenko, A.S., Patterson, L.B., Sood, V.D., Nudel, U., Yaffe, D., Baker, D., and Ruohola-Baker, H. (2006) Dissecting Muscle and Neuronal Disorders in Drosophila Muscular Dystrophy, submitted.
Gray, E.E., Yatsenko, A.S., Shcherbata, H.R., Patterson, L.B., Sood, V., Baker, D., and Ruohola-Baker, H. (2006) A putative SH3-domain binding motif but not the C-terminal Dystrophin WW-domain binding motif is required for Dystroglycan function in cellular polarity, submitted.
Hatfield SD, Shcherbata HR, Fischer KA, Nakahara K, Carthew RW, Ruohola-Baker H. Stem cell division is regulated by the microRNA pathway. Nature. 2005 Jun 16;435(7044):974-8.
Althauser C, Jordan KC, Deng WM, Ruohola-Baker H. Fringe-dependent notch activation and tramtrack function are required for specification of the polar cells in Drosophila oogenesis. Dev Dyn. 2005 Apr;232(4):1013-20. PMID: 15765546
Jordan KC, Hatfield SD, Tworoger M, Ward EJ, Fischer KA, Bowers S, Ruohola-Baker H. Genome wide analysis of transcript levels after perturbation of the EGFR pathway in the Drosophila ovary. Dev Dyn. 2005 Mar;232(3):709-24.
Shcherbata HR, Althauser C, Findley SD, Ruohola-Baker H. The mitotic-to- endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions. Development. 2004 Jul;131(13):3169-81.
Schaeffer V, Althauser C, Shcherbata HR, Deng WM, Ruohola-Baker H. Notch-dependent Fizzy-related/Hec1/Cdh1 expression is required for the mitotic-to-endocycle transition in Drosophila follicle cells. Curr Biol. 2004 Apr 6;14(7):630-6.
Findley, S., Tamanaha, M., Clegg, N., and Ruohola-Baker, H. (2003) Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/Ago1 homolog, Aubergine, in Nuage. Development 130: 859-871.
Deng WM, Schneider M, Frock R, Castillejo-Lopez C, Gaman EA, Baumgartner S, Ruohola-Baker H. (2003) Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila. Development.130:173-184.
Deng, W-M, Althauser, C. and Ruohola-Baker, H. (2001) Notch-Delta signaling induces a transition from mitotic cell cycle to endocycle in Drosophila follicle cells, Development, 128:4737-4746
Clegg, N., Findley, S., Mahowald, A.P., and Ruohola-Baker, H. (2001) Maelstrom is required to position the MTOC in stage 2-6 Drosophila oocytes. Dev Genes Evol. 211:44-48.
Jordan, K., Clegg, N., Blasi, J., Morimoto, A., Sen, J., Stein, D. McNeill, H., Deng, W-M., Tworoger, M. and Ruohola-Baker, H. (2000) The homeobox gene mirror links EGF signaling to embryonic dorso- ventral axis formation through Notch activation. Nature Genetics, 24, 429-433.
Deng, W.-M.and Ruohola-Baker, H. (2000) Laminin A is required for follicle cell-oocyte signaling that leads to establishment of the anterior-posterior axis in Drosophila. Curr Biology 10, 683-686.
Bryant,Z, Subrahnmanyan,L, LaTray,L., Tworoger,M., Liu,C- R., Li,M-J, van den Engh, G., and Ruohola-Baker, H. (1999) Characterization of differentially expressed genes in purified Drosophila follicle cells: towards a general strategy for cell type-specific developmental analysis. Proc Natl Acad Sci U S A. 96, 5559-5564.
Larkin,M.K., Deng,W-M, Tworoger,M., Holder,K., and Ruohola-Baker, H. (1999) Role of Notch pathway in terminal follicle cell differentiation in Drosophila oogenesis. DGE,209, 301-311.
Tworoger,M., Larkin,M.K., Bryant,Z., and Ruohola-Baker, H. (1998) Mosaic analysis in the Drosophila ovary reveals a common, Hedgehog inducible precursor stage for stalk and polar cells. Genetics, 151, 739-748.
Clegg,N.J., Frost,D.M., Larkin,M.K., Subrahmanyan,L., Bryant,Z., and Ruohola-Baker,H. (1997) maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA. Development, 124, 4661-4671.
Morimoto,A.M., Jordan,K.C., Tietze,K., Britton,J.S., O'Neill,E.M., and Ruohola-Baker, H. (1996) Pointed, an ETS domain transcription factor, negatively regulates the EGF receptor pathway in Drosophila oogenesis. Development 122, 3745-3754.
Larkin, M.K., Holder, K., Yost,C., Giniger,E., and Ruohola-Baker, H. (1996) Expression of constitutively active Notch arrests follicle cells at a precursor stage during Drosophila oogenesis and disrupts the anterior-posterior axis of the oocyte. Development 122, 3639-3650.
Ruohola-Baker, H., Jan,L.Y., and Jan,Y.N. (1994) The role of gene cassettes in axis formation during Drosophila oogenesis. Trends in Genetics, 10, 89-94
Clark,I., Giniger,E., Ruohola-Baker, H., Jan,L.Y., and Jan,Y.N. (1994) Microtubule polarity provides an anteriorposterior axis to the Drosophila oocyte. Current Biology 4, 289-300.
Ruohola-Baker,H., Grell,E., Baker,D., Jan,L.Y., and Jan,Y.N. (1993) Spatially localized rhomboid is required for establishment of the dorsal-ventral axis in Drosophila oogenesis. Cell 73, 953-965
Ruohola,H., Bremer,K.A., Baker,D., Swedlowe,J.R., Jan,L., and Jan,Y.N. (1991) Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila. Cell 66, 433-449