Using Axon Regeneration in Drosophila to Understand

Signaling and Intracellular Organization of Neurons

David F. Lee, Joseph Kim, Matthew Shorey, Alex Weiner, Melissa M. Rolls
Penn State Department of Biochemistry and Molecular Biology
STAT 92 E level increases post axon injury

Abstract

Hypotheses

Axon regeneration in Drosophila neurons has been studied

immensely and is becoming to be better understood. Though the
DLK signaling pathway initiates axon regeneration, it is not sufficient.
Other signaling pathways might be involved along with the DLK
pathway. The STAT pathway is known to play a major role in
mammalian-injury regeneration, and we can use Drosophila to
determine whether STAT acts downstream of DLK or acts
independently. The hypothesis for this topic is that the STAT
pathway plays a key role in axon regeneration and runs parallel to the
DLK pathway. To test this, we are able to knock down DLK and
observe STATs roles in axon regeneration. At the same time of
investigating the signaling pathways that initiate regeneration, we can
use live cell imaging to observe changes in the cell. Ribosomes,
which are a major player in a cells response to injury, accumulate in
the dendrites, but not the axon of Drosophila neurons. By cutting the
axon, it has been proven that a preexisting dendrite will flip its polarity
and morphologically extend out in roughly 48 hours, becoming the
cells new axon. This leaves an ideal model, which is dendritic
branches that come off the newly formed axon. It is hypothesized that
motor proteins traveling along minus- end-out microtubules are
responsible for trafficking these ribosomes. A drop in ribosomal
concentration in the newly formed axon and the dendrites coming off
this axon would support this hypothesis.

1. The JAK/STAT signaling pathway is expected to be

involved in axon regeneration.

2. The JAK/STAT signaling pathway runs independently

from the DLK pathway.
3. The trafficking of ribosomes is expected to occur
through motor proteins that travel along microtubules
that exhibit minus-end-out polarity.
a. Dyneins travel along minus-end-out microtubules.
b. If dyneins were responsible for trafficking ribosomes,
this would help explain why ribosomes are found in
dendrites, but not axons.

Procedure

RTNL2 RNAi on
2 33320

Stat 92E GFP, 221 Gal4, EB1-RFP

TM6

Green intensity represents the expression of STAT 92E.

STAT 92E level elevates after the cut.

The following crosses were established to test the first two

hypotheses. The axons of DDAE neurons were cut with the
UV pulse laser.

Accumulation of Ribosomes in Dendritic

Branch-Points

Introduction
Drosophila melanogaster are used as a model system for the peripheral
nervous system. UAS-GAL4 and end binding protein 1(EB1) allows us
to visualize neuronal morphology.

x
UAS-GAL4 X EB1 tester line

JAK/STAT signaling pathway mechanism

Method of Action for High-Powered Microscope

1. Image the pre-cut neuron.
2. Cut axon with the UV pulse laser.
3. Re-image the neuron certain hours
after the cut.
4. Compare the cellular organizations.

To examine the trafficking of ribosomes after the cut, the

intensity of ribosomes can be quantified and observed any
changes in concentration in the dendritic branch points and
the dendritic branch which is converted to the new axon.

Knocking down STAT 92E gene suppresses

axon regeneration of ddaE neuron
Regeneration in the Wild Type Control

221
Gal4,
EB1-RFP
221 Gal4 UAS.YL-10 on 3 x
TM6
YFP-tagged L-10 protein allows ribosomal visualization.
An intensity drop in axonal dendrites may suggest that
dyneins are responsible for ribosomal trafficking.

Future Directions
1.Quantification of the tip growth in the control
and STAT 92E RNAi.

JAK/STAT pathway has been proven to be involved

in mammalian neurological regenerations, and it is
hypothesized to be involved in axon regeneration.

2. Crossing other JAK/STAT RNAi lines against

EB1 tester line.

Dendrite converted to Axon post axotomy

3. Examine if JAK/STAT is independent from the

DLK pathway.
a. JAK/STAT RNAi Lines x PUC-GFP reporter
RTNL2 RNAi on 2
33320

UAS-Gal4, 221 EB1-GFP

TM6

Regeneration in the STAT RNAi assay

b. DLK RNAi Lines x STAT-GFP reporter

4. Quantification of ribosomal intensity in pre-cut

and 72 hour post-cut neurons.
5. Comparison of data to rare, naturally-occuring
axonal dendrites.

Acknowledgements
STAT 92E RNAi

Axotomy causes flip in microtubule polarity, converting minusend-out dendrite to plus-end-out axon.
This leaves axonal dendrites.

UAS-Gal4, 221 EB1-GFP

TM6

The length of regeneration observed in an assay

with STAT protein knock-down is much shorter
than that of the control.

The Rolls Lab

Biochemistry and Molecular Biology
Department of the Eberly College of Science
The National Institute of Neurological Disorders and Stroke