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.

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2. Most ina-1(gm39) mutants have ACh and GABAergic axon pattern defects!

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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

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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

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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

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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.

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2. Abnormal axonal projection in ina-1(gm39) mutants

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B"

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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.

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ina-1(gm144) mutantC" D"

Cholinergic!!GABAergic

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B.

ina- 1 (gm144) mutant with wildtype INA-1

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Percen

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Axon

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CONCLUSIONS

Diagram!taken!from:!h<p://www.kAstate.edu/hermanlab/!