Symposium 2015 FINAL
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Transcript of Symposium 2015 FINAL
Examination of ina-1 Mutants in Caenorhabditis Elegans to Better Understand Axonal Patterning Priya Ahluwalia, Mary Gonring, Cassie Lincoln, M.L. Lemons
Department of Natural Sciences, Assumption College, Worcester MA 01609
1. INA-1 is expressed in all GABAergic neurons.2. Most ina-1 (gm39) mutants have axonal errors.3. ina- 1 (gm144) mutant with wildtype INA-1 shows many misprojected axons suggesting INA-1 may not act cell autonomously in GABAergic neurons.
L1
2. Most ina-1(gm39) mutants have ACh and GABAergic axon pattern defects!
30Percent growth cone collapseE.Netrin-1 Ab0102030Percent growth cone collapseNetrin-1.
3. Preliminary data suggests that INA-1 may not work cell autonomously!
Percentage of worms with cholinergic (ACH, green) or GABA (red) axonal errors in ina-1(gm39) (N=15). j
INTRODUCTION
Worm strains: Bristol N2 Caenorhabditis elegans were used for this studies . The following strains were used :ufis34;visis48, ufis34 (unc-47::mCherry); vsis48 (unc-17::GFP); ina-1 (gm144); ufis34;visis48 ina-1 (gm39); ufis34;vsis48, gmIs5 (INA-1::GFP), ufex530 (unc-47::ina-1, unc-47::mCherry, lgc::GFP) ; oxis-12 (unc-47::GFP).
MATERIALS AND METHODS
BACKGROUND
30Percent growth cone collapseE.Netrin-1 Ab0102030Percent growth cone collapseNetrin-1
A.
C.elegans as a model to study neuronal growth
A. Image of the 1 mm nematode Caenorhabditis elegans.. B. Cartoon of integrin molecule with an alpha subunit (purple) and a beta subunit (orange). The carton on the left represents an inactivated integrin and the image on the right is an integrin in the activated, high ligand affinity state.
RESULTS
30Percent growth cone collapseE.Netrin-1 Ab0102030Percent growth cone collapseNetrin-1
Figure 1. Fluorescent images of INA-1 (green) and GABA neurons (red) in vivo. Neuronal cell bodies (that appear as ovals) are located in the ventral nerve cord.
1. INA-1 is expressed in GABAergic Neurons!
INA-1::GFP
GABA
Merge
30Percent growth cone collapseE.Netrin-1 Ab0102030Percent growth cone collapseNetrin-1.
Future DirectionsFigure 4: Inactive and active integrin. PAT-3, a beta subunit which causes an integrin to be constantly active. The future plan is to make a pat-3 transgenic mutant that has constant expression of PAT-3.
30Percent growth cone collapseE.Netrin-1 Ab0102030Percent growth cone collapseNetrin-1.
2. Abnormal axonal projection in ina-1(gm39) mutants
A."
B"
30Percent growth cone collapseE.Netrin-1 Ab0102030Percent growth cone collapseNetrin-1
The ability of neurons to properly extend axons during development by a complex extracellular milieu and ultimately reach their appropriate targets is remarkable. The molecular mechanisms that drive this impressive navigational feat are not yet fully understood. Previous studies suggest that a family of transmembrane heterodimeric proteins, called integrins, play an important role in neuronal motility. A functional integrin has two subunits: 1) an alpha subunit, such as INA-1 and 2) a beta subunit, such as PAT-3. We chose to more fully characterize the effects of integrins on axon patterning in the genetically-powerful model organism Caenorhabditis elegans (C.elegans). The focus of our study was to: 1) determine which axons express INA-1, 2) better understand the role of integrins in axonal patterning and 3) study if INA-1 works cell autonomously in GABA motor neurons. The future direction of our study will be to better classify the role of integrin activation (a conformational change to a high ligand affinity state) on axon guidance.
C"
A"
A. Image of GABA (red) and cholinergic (green) neurons in an ina-1(gm39) mutant. Neuron cell bodies are located in the ventral nerve cord (bottom). Axons project from the ventral nerve cord to the dorsal nerve cord. B. Higher magnification of a wild type axon. Note the straight projection from ventral nerve cord to dorsal nerve cord. C. Image of ina-1(gm39) mutant worm with abnormal projection, as indicated by arrows.
Wildtype ina-1(gm39) mutant
GABAergic neuron GABAergic!neurons!
ina-1(gm39) mutantina- 1 (gm144) mutant with wildtype INA-1A"
B"
Ina-1 (gm144) mutant
ina- 1 (gm144) mutant with wildtype INA-1
Ina-1 (gm144) mutant
A. Image of GABA (red) and GABA (green) neurons in ina- 1 (gm144) mutant with wildtype INA-1 worm. B. Image of the same worm as A GABA (red) neurons in ina- 1 (gm144) mutant with wildtype INA-1 worm. C. Abnormal axon projection in ina-1(gm144) mutant, as indicated by arrows. D. Axon misprojection in ina- 1 (gm144) mutant with wildtype INA-1, as indicated by arrows.
B"
ina-1(gm144) mutantC" D"
Cholinergic!!GABAergic
A"
B.
ina- 1 (gm144) mutant with wildtype INA-1
0!10!20!30!40!50!60!70!80!90!
100!
Percen
tage"of"W
orms"W
ith"
Axon
al"Errors"(%)"
CONCLUSIONS
Diagram!taken!from:!h<p://www.kAstate.edu/hermanlab/!