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Full Abstracts – NEURAL DEVELOPMENT
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32
Dopaminergic precursor fate is established by
gsb-n
and
slp
at the intersection of Wg and Hh signaling.
Joseph D. Watson, Stephanie B. Stagg,
Stephen T. Crews. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill.
Our lab is focused on identifying the transcription factors that specify the dopaminergic H-cell from a pool of Midline Precursors (MPs). A screen of
candidate genes expressed in the midline revealed that proper specification of dopaminergic fate required the paired homeobox gene
gooseberry-neuro
(
gsb-
n
). Loss of
gsb-n
eliminated the gene that encodes tyrosine hydroxylase (
pale
) from the midline CNS, while ectopic expression of
gsb-n
generated additional
dopaminergic neurons. In both cases, the specification of MP1 midline neurons was disrupted. Closer examination revealed that the loss of
gsb-n
generated
additional MP1 neurons, while misexpression of
gsb-n
duplicated MP3 at the expense of the MP1 precursor. However, misexpression of
gsb-n
had only a
mild effect on posterior MPs. These data suggest that transcription factors arrayed along the A-P axis spatially restrict dopaminergic specification to the
anterior of each segment. We showed that the fork-head transcription factor
sloppy-paired
(
slp
) was expressed exclusively in MP1 and MP3. This expression
required
wingless
, and mutations in
slp
resulted in additional posterior MPs and loss of MP1 and MP3. This suggests that
slp
segregates anterior MPs from
posterior MPs. Thus, MP1 and MP3 initially have a common transcriptional program. What differentiates MP3 from MP1? We showed that ectopic
expression of
hedgehog
throughout the midline led to the generation of two H-cells and the loss of MP1 neurons. Consistent with these results,
gsb-n
was
ectopically activated in MP1s. We also showed that the transcription factors
lethal of scute
,
tup
, and
BarH1
act downstream of
gsb-n
to activate a
dopaminergic program in H-cell. Finally, we observed similar cell fate changes in non-midline lateral CNS derived dopaminergic neurons in each of these
mutant backgrounds. Thus, our data suggest that a common set of transcriptional programs specify dopaminergic precursor fate in the Drosophila CNS.
33
Combinatorial input from two spatial axes generates neuronal diversity in the
Drosophila
medulla.
Ted Erclik, Xin Li, Claire Bertet, June Ng, Claude
Desplan. Department of Biology, New York University, New York, NY, USA.
The
Drosophila
medulla is the largest neuropil in the optic lobe and is responsible for the processing of both color and motion detection signals. While
over 70 neuronal cell types have been identified in the medulla, little is known about how these neurons are specified. We have performed a transcription
factor antibody screen in the medulla and have identified 35 genes that are expressed in subsets of medulla progenitors and neurons. By mapping these
transcription factors onto the larval optic proliferation center, the structure from which the adult medulla is generated, we find that neuronal specification in
the medulla is the product of the intersection of two spatial axes: (1) in the medio-lateral axis, neuroblasts switch between five distinct fates to generate
neurons of different identities. As they age, neuroblasts first express Homothorax (Hth), then Eyeless, followed by Sloppy-paired 1, Dichaete and, finally,
Tailless. We find that later neuroblast genes repress the expression of earlier ones; (2) in the dorsal-ventral axis, the neuroepithelial crescent from which the
neuroblasts are generated is sub-divided into dorsal (Optix), central (Vsx1) and ventral (Optix+Hedgehog) compartments. While the sequential progression
of neuroblasts is identical in each region, the types of neurons that are generated by a given neuroblast are region-specific. We have determined how these
two axes intersect to generate diversity in the progeny of Hth neuroblasts. Hth neuroblasts generate Mi1 neurons throughout the larval crescent but Pm3
neurons specifically in the center. In these central neuroblasts, Vsx1 is co-expressed with Hth where it directs the formation of Pm3s;
vsx1
is required for the
expression of the Pm3 markers Seven-up and Prospero but not the Mi1 marker Bsh. Thus, Pm3 specification is the product of combinatorial input from both
the neuroblast (Hth) and dorsal-ventral (Vsx1) axes. We have extended this analysis to the other neuroblasts and have mapped 11 additional neuronal-types
to distinct regions in the larval optic proliferation centers.
34
Slit/Robo-mediated axon guidance in
Tribolium
and
Drosophila
: divergent genetic programs build insect nervous systems.
Tim Evans, Greg Bashaw.
Dept Neuroscience, Univ Pennsylvania Sch Med, Philadelphia, PA.
Roundabout (Robo) family receptors control diverse axon guidance decisions during nervous system development in bilaterian animals. In
Drosophila
,
Robo and Robo2 mediate midline repulsion in response to Slit, while Robo2 and Robo3 specify the lateral position of longitudinal axon pathways. Alone
among the fly Robos, Robo2 can also promote midline crossing. Notably,
Drosophila
robo2 and robo3 are products of a recent gene duplication, suggesting
that their distinct roles in controlling midline crossing and promoting lateral and intermediate pathway formation may be recent evolutionary developments.
To gain insight into the evolution of axon guidance receptor functions in insects, we have characterized Slit/Robo-mediated axon guidance in the flour beetle
Tribolium castaneum
, which unlike
Drosophila
has only two Robo receptors: Robo (TcRobo) and the ancestor of Robo2 and Robo3 (TcRobo2/3). Using
RNAi, we show that
TcSlit
is required for midline repulsion of axons in the beetle embryonic CNS, and that both
Tribolium
Robos contribute to this activity.
Longitudinal axon pathways in the
Tribolium
embryonic CNS form in distinct medial, intermediate, and lateral zones, despite the presence of only two
Robos. Individual knockdowns demonstrate that beetle Robos have specialized axon guidance functions:
TcRobo
is dedicated to midline repulsion, while
TcRobo2/3
also regulates longitudinal pathway formation.
TcRobo2/3
knockdown mimics aspects of both
Drosophila
robo2 and robo3 mutants, suggesting
that these two genes in flies share a role that is performed by a single ancestral gene in other insects. In addition, TcRobo2/3 is unable to promote midline
crossing of
Drosophila
axons, suggesting that the pro-crossing role of fly Robo2 arose after its divergence from Robo3. Together, our results suggest that the
functional diversification of Robo receptors during insect evolution has involved gene duplication and both loss and gain of axon guidance activities, and
reveal that modern insects deploy divergent genetic programs to control equivalent axon guidance decisions during development.