Differentiation of dendrites and the analysis of spine- like structures on Lobula Plate Tangential Cells in Drosophila melanogaster

The development of dendrites leads to the establishment of
cell-type specific morphology of dendritic trees that eventually
determines the way in which synaptic information is processed
within the nervous system. The aim of this study was to investigate
dendritogenesis of Drosophila motion-sensitive Lobula Plate
Tangential Cells (LPTCs) and to understand the role of cytoskeletal
molecules in these developmental processes. I employed genetic
techniques to obtain fluorescent labeling exclusively in the
neurons of interest. In order to visualize the LPTCs confocal
imaging was applied. Time point analysis allowed me to follow and
describe the phases of LPTC differentiation in the intact
Drosophila brain starting from the third instar larva throughout
the pupal stages until adulthood. I determined the time when the
initial growth of LPTC dendrites starts and showed it to be
directional from the beginning. Additionally, I demonstrated that
the phase of extensive dendritic growth and branching precedes
reorganization processes that lead to establishment of the final
architecture of LPTC dendritic trees. In parallel, I attempted to
analyze the contribution of actin and tubulin in the shaping of the
neurons. In these experiments actin-GFP localized to dendritic
termini whereas tubulin-GFP was mainly observed in the primary
dendritic branches. These data showed clear similarities between
the cytoskeletal organization of LPTCs dendrites and vertebrate
neurons. The discovery of the actin enrichment in dendritic termini
made me conduct a set of experiments to test if these protrusions
are the counterparts of vertebrate spines. I performed a thorough
quantitative analysis of spine- like protrusions present on LPTC
dendrites. Morphological features like the density and shape of the
LPTC spine- like protrusions appeared to be comparable to
hippocampal spines. Using immunohistochemical methods I
demonstrated that LPTC spine-like protrusions are sites of synaptic
contacts. The ultrastructural analysis supported the
immunohistochemical data and showed that synaptic transmission
takes place at the LPTC spine-like protrusions. Next, I tried to
genetically modify these structures by generating LPTC mutant for
genes which have vertebrate homologues known to alter spine
morphology. I showed that dRac1 can modulate significantly the LPTC
spine-like structure density. Finally, I tried to check if
Drosophila LPTC spine-like structures are motile. To conclude, I
showed an initial description of LPTC dendritogenesis and the
subcellular localization of actin and tubulin in these neurons. The
actin enriched spine-like structures detected on the LPTC dendrites
are sites of synaptic contacts, thus resemble vertebrate spines.