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Full Abstracts – NEURAL DEVELOPMENT
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35
Axonal branching and synaptic connectivity of mechanosensory axons.
Derya Ayaz, Dan Dascenco, Olivier Urwyler, Dietmar Schmucker. Vesalius
Research Center, VIB, Leuven, BELGIUM.
During development neurons are precisely organized to connect with each other and give rise to the complex pattern of the nervous system. The wiring
complexity is highly increased by the ability of a single neuron to connect with multiple target neurons by forming specific axonal and dendritic branches. In
contrast to research on axon guidance, much less is known about basic mechanisms underlying spatially restricted axon branching. How
axon branching
is
induced molecularly and how external stimuli influence this process
remains largely elusive
. We have established an
in vivo
system
for studying axon
branching using mechanosensory neurons that exhibit an invariant genetically hardwired branching morphology in the ventral nerve cord. We will present a
time-lapse
in vivo
analysis of axon branching of afferent projections within the pupal CNS. Our analysis reconstructs the developmental sequence of sensory
axon branching and document for the first time growth cone dynamics, spatially restricted axonal sprouting, and interstitial branch formation within the
pupal CNS. Moreover we will present a systematic genetic analysis of key regulators of axon branching. Currently we are focusing on a novel kinase,
distantly related to STE-20 like kinases, which is essential for sensory axon branching. Based on this we will discuss a new molecular pathway that
selectively controls axonal branching of mechanosensory neurons.
36
Defining microRNA function during synapse development using the Drosophila microRNA sponge.
Elizabeth M. McNeill, Carlos Loya, Tudor Fulga,
David Van Vactor. Cell Biology, Harvard Medical School, Boston, MA.
MicroRNAs (miRNAs) have emerged as key candidates to regulate the deployment of proteins that control synaptic morphogenesis and function.
However, our understanding of the structural, physiological and cellular mechanisms by which miRNAs control synapse development remains relatively
unexplored. We have generated a library of Drosophila miRNA sponge (miR-SP) transgenics, which we have screened to determine the effects of miRNA
depletion to synapse structure. This screen has confirmed many of the previously known miRNA players at the synapse as well as begun to identify some
unique miRNAs involved in synaptic morphogenesis. Using a miR-SP targeting the conserved miRNA, miR-8, we have discovered that although expressed
on both sides of the synapse, miR-8 is an essential postsynaptic regulator of neuromuscular junction (NMJ) differentiation, subsynaptic reticulum
elaboration, and physiology. A key to this postsynaptic-specific regulation of NMJ development is the miR-8 target gene Enabled (Ena), a well-established
regulator of actin dynamics. Using a combination of microarray, in silico, genetic and biochemical tools, we find that muscle-specific repression of Ena
accounts for most of miR-8’s potent regulation of NMJ ultrastructure and morphogenesis. Moreover, structural mutant analysis of the conserved domains in
mammalian Ena (Mena) suggest that the C-terminal Ena/VASP homology 2 (EVH2) domain, which is comprised of a G-actin binding, F-actin binding and
coiled coil motif, is both necessary and sufficient for postsynaptic Ena localization and function. Consistent with the known antagonism between Ena/VASP
proteins and actin capping proteins in F-actin dynamics at the molecular level, we see a partial rescue of Ena overexpression with capping protein beta gain-
of-function at the synapse. Our study reveals a tissue-specific function for miR-8 in synapse formation through the control of the postsynaptic actin
cytomatrix, and provides new insight into the regulatory repertoire of miRNA in nervous system development.
37
Nucleotide sugar transporter Meigo regulates both dendrite and axon targeting of synaptic partners through Ephrin signaling in the olfactory
system.
Sayaka Sekine
1
, Liang Liang
6
, Miki Yamamoto-Hino
4
, Satoshi Goto
4
, Hideyuki Okano
4
, Liqun Luo
5,6
, Masayuki Miura
1,2
, Takahiro Chihara
1,3
. 1)
Genetics, Grad Sch Pharm Sci, Univ. Tokyo, Japan; 2) CREST; 3) PRESTO; 4) Dept. Physiol, Keio Univ, Tokyo, Japan; 5) HHMI; 6) Dept. Biol. Stanford
Univ, CA.
The wiring of neural network results from accurate synaptic matching enabled by precise targeting of axons and dendrites. During the development of
olfactory system, the axons of each first-order olfactory receptor neuron (ORN) and the dendrites of each second-order projection neuron (PN) target one of
~50 glomeruli in the antennal lobe (AL), resulting in one-to-one connections of proper synaptic partners. To elucidate the mechanism of dendrite targeting of
PNs, we performed MARCM-based genetic screen and isolated a mutant,
meigo
(
medial glomeruli
). The dendrite of
meigo
-/-
PN spilled over from destined
glomeruli, and mistargeted to the medial side of the AL. Interestingly, axon targeting of
meigo
-/-
ORNs was also medially shifted in the AL. The responsible
gene (
meigo
) encodes a nucleotide sugar transporter that is located at ER. These results suggest that Meigo is cell-autonomously required in synaptic
partners for neuronal targeting along the mediolateral axis in the AL, probably by regulating the glycosylation of cell surface proteins. To identify the cell
surface proteins, we performed genetic modifier screen and found that overexpression of
ephrin
in
meigo
-/-
PNs significantly suppresses the dendrite
mistargeting phenotype. Downregulation of
ephrin
in PNs exhibited the dendrite spillover, which is similar to that in
meigo
-/-
PNs. In contrast to medially
shifted dendrites in
meigo
-/-
PNs, overexpression of
ephrin
caused dendrite mistargeting to the lateral side of the AL. Moreover, biochemical and genetic
analyses revealed that efficient
N
-glycosylation of Ephrin requires Meigo in S2 cells, and Ephrin
N
-glycosylation is essential for its full function in neuronal
targeting
in vivo
. Thus,
meigo
regulates neuronal targeting of synaptic partners in the AL through facilitation of Ephrin signaling partly by
N
-glycosylation.