Imaging niche signals in the Drosophila germ line
Previous and Current Research
Stem cells are essential for tissue formation during development, and allow tissue maintenance and repair during the adult life of an organism. However, stem cell identity, survival, and activity are not intrinsic properties that stem cells possess once they are specified. Instead, these properties are in many cases induced and maintained by a local signalling microenvironment termed niche. We are interested in how the intercellular communication within a niche governs stem cell function at the molecular, subcellular, and tissue level, and use the gonadal stem cell niche of the fruit fly Drosophila as a model system.
Our key questions are i) how tissue organization controls the range of the niche signals and therefore niche size, ii) how the various signalling pathways controlling stem cell behaviour are integrated, and iii) how regulation of the signalling pathways is implememented at the cell biological level.
In the fly testis, germline stem cell (GSC) fate is maintained through a signal by the BMP family growth factor Dpp that is emanating from the adjacent somatic niche cells. Interestingly, only GSCs in immediate contact with the signal producing somatic cells are able to receive this niche signal, although in other contexts BMPs act as archetypal long range signalling factors.
To study how this range restriction is achieved we have developed a a fluorescent reporter for BMP receptor activation that allows us to track niche signalling live and with subcellular resolution. Our reporter is based on a conformationally sensitive GFP derivative fused to the Drosophila type I BMP receptor Thickveins. In the inactive state, its fluorescence is switched off, while receptor activation allows the GFP moiety to adopt a fluorescent conformation. We could thus show that transduction of the niche signal is tightly confined to adherens junctions between niche cells and GSCs. This synapse-like arrangement limits the ability of the ligand to spread through the tissue.
We have since started to adapt our reporter technology to image the activation state of components of other signalling pathways, e.g. the Hh and Jak/Stat pathways. While we could show that Hh is an essential niche factor for the somatic stem cells in the testis, much less is known at the cell biological level about how the Hh signal is transduced. In the absence of ligand, the Hh receptor Patched (Ptc) inhibits the key signal transducer Smoothened (Smo), retaining Smo on intracellular membranes in a nonphosphorylated state. Upon Hh binding, Ptc is inactivated, and Smo relocalizes the plasma membrane, becomes phosphorylated, and starts to form oligomeric clusters. We have therefore generated a fluorescence based sensor for Smo phosphorylation. In combination with mathematical modelling we could thus show that translocation of Smo to the plasma membrane is sufficient for pathway activation. Smo localization therefore acts upstream of Smo phosphorylation, and is thus presumably the cell biological process directly targeted by Ptc.
In addition, we have started to study these signalling pathways with the help of biophysical tools such as fluorescence lifetime imaging / Förster resonant energy transfer (FLIM/FRET) microscopy and fluorescence cross-correlation spectroscopy (FCCS), addressing questions like ligand-receptor affinities or the clustering of receptors and other signalling molecules within their native environment of living cells.
We could thus e.g. show that – in contrast to the textbook model – Smo clustering in response to Hh does not require phosphorylation of the cytoplasmic tail.
Future Projects and Goals
In the medium term we want to understand how a model stem cell niche works at the molecular and tissue level, to the extent that that we will be able to build a quantitative model of all communication events and cellular interactions that govern niche function. Since stem cell culture is still an unresolved problem in regenerative medicine this should be of immediate interest beyond the field of fly developmental biology. In addition, many of these signalling pathways are also involved in clinically relevant diseases such as asthma that are unrelated to stem cells. Our cell biological and biophysical insights into these signal transduction machineries can therefore provide novel entry points for their pharmacological modulation.
Methodological and Technical Expertise
- Drosophila genetics and cell biology,
- confocal imaging,
- live cell imaging,
- live cell fluorescence cross-correlation spectroscopy (FCCS),
- FLIM/FRET microscopy
Kupinski, AP, Raabe, I, Michel, M, Ail, D, Brusch, L, Weidemann, T, and Bökel, C.
Visualizing Smoothened phosphorylation and clustering in vivo.
J Cell Sci, in press (2013)
Bökel, C, Brand, M.
Generation and interpretation of FGF morphogen gradients in vertebrates.
Curr Op Gen Dev, doi: 10.1016/j.gde.2013.03.002. (shared corresponding authorship) (2013).
Michel, M., Kupinski, A. P., Raabe, I. and Bökel, C.
Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche
Development 139(15): 2663-9. (2012)
Michel, M., Raabe, I., Kupinski, A. P., Perez-Palencia, R. and Bökel, C.
Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche
Nat Commun 2: 415. (2011)
Weidemann, T., Worch, R., Kurgonaite, K., Hintersteiner, M., Bökel, C. and Schwille, P.
Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits
Biophys J 101(10): 2360-9. (2011)