C. Elegans: Olfaction and chemotaxis

Introduction - Orientation with C. *elegans* and its life cycle

* *C. elegans* is a free-living worm that feeds on soil bacteria.
* These worms grow quickly, developing from embryo to adult in three days.
* These organisms are often studied in development and genetic studies since they are transparent, their genome has been sequenced and annotated and they are easily manipulated.

Introduction - Chemotaxis assay

* Many organisms use chemotaxis to an external site to **seek out** food sources, **avoid** noxious substances, and **find** mates.
* Assays have even been developed to demonstrate the ability of *C. elegans* to store short and long term memory by showing that associations are made by the worms between chemoattractants and food (OP50).

Introduction - Chemosensory neuron signaling

* Adult *C. elegans* have only 302 neurons. Of these, 32 (16 pairs) have been identified as chemosensory neurons. Chemosensory neurons are found in the amphid, phasmid, and labial organs. These neurons contain cilia that are exposed to the environment via socket or sheath cells.

LacZ staining

* One type of modification that can be made to study expression patterns of genes is the insertion of reporter genes.
* The LacZ gene codes for the enzyme β-galactosidase (β-gal). β-gal can be used as a reporter because when it cleaves the substrate X-gal (5-bromo-4-chloro-3-indolyl-b -galactopyranoside), a blue precipitate is produced.

Olfaction

C. *elegans* is a useful model organisms for…

studying olfaction.

* Similar sensory neuron.
* Similar receptor and intracellular signaling mechanism.
* Easy to assay response; chemotaxis.

Smelling with cilia

Chemotaxis

* Movement of a cell or organism towards or away from a chemical.
* C. *elegans* does this to find food or a hospitable environment.
* This is a convenient behavior that can be used to assess the function of chemosensory neurons.

Chemotaxis assay

* Chemoattractant today is diacetyl, which is a byproduct of fermentation.
* Binds to the ODR-10 receptor, which is localized to AWA neurons in the C. *elegans* sensory organ.

ORD-10 and chemotaxis

* ORD-10 is a G protein coupled receptor (GPCR).
* GPCRs interact with G proteins.
* Generic GPCR pathway at right.

Assay

* Notice the precise layout of the plate.
* Necessary to set up uniform gradients of chemical.
* Must was worms several times.
* **Bacteria will compete.**
* Spot worms and attractant the same way from plate to plate.

Analysis

Why this assay is useful

* Easy to score test of chemotaxis.
* Involves sensing gradients of chemicals, triggering a behavior and the ability to carry out that behavior.
* Can be used to characterize mutants or normal neuronal development.
* Identifying mutants that can’t chemotax can be used to identify important genes and proteins.
* This is one of the ways to discover how the system works.
* Can be used to test whether different compounds are chemoattractants.

Procedure for day one

* Identify different phases of the life cycle and compare lines A and B.
* Plate and wet mount.
* Take pictures. Note size.
* Set up chemotaxis assay to diacetly.
* Lots of washes are required, so please try to coordinate with other groups when using the centrifuge!
* Don’t spot the chemoattractant until the worms are spotted!
* Worms need to migrate for one hour.
* Plates will be stored at 4°C until the next class and scored then.

Reporter genes

Want to know something about the regulatory region (maybe when the gene would be turned on or where).

Does not have to be GFP

* In our transgenic strains of C. *elegans*, the lacZ gene is fused to the promoter of either:
* gpa-1: to stain sensory neurons.
* unc-54: to stain body wall muscles.

LacZ/Beta-gal staining

* When the gene (in our case: unc-54 or gpa1) is expressed, so is lacZ.
* The lacZ gene codes for b-gal.
* Beta-gal is an enzyme that cleaves the substrate X-gal and produces a blue color.
* Any blue color in the worm indicates that the reporter gene is expressed in that cell.

Examples of X-gal marking gene expression in C. *elegans*.

Procedure

* Score Chemotaxis Assay from last lab.
* LacZ Staining: neurons, muscles, and wild type strains.
* Add 1 mL of water to each plate and transfer to a 1.5 mL centrifuge tube.
* Pellet by centrifuging for 1 min at 400xg.
* Remove water.
* Add 1 mL of water to tube and resuspend pellet by inverting tube.
* Centrifuge again to pellet worms.
* Remove water.
* Freeze by submerging in liquid nitrogen (BE CAREFUL: Must wear safety glasses!!).
* Suspend worms in 250 uL of ice cold acetone and incubate on ice for 3 minutes.
* Remove as much of acetone as possible.
* Suspend worms in 200 uL of staining solution.
* Give tube to TA to keep at 37 degrees overnight.

Sensory neuron staining

* To visualize sensory neurons we used a line of worms that express the lacZ gene under the control of the gpa-1 promoter.
* gpa-1:
* Encodes a member of the G protein alpha subunit family of heterotrimeric GTPases that is involved in chemotaxis to water-soluble odorants.
* It is expressed in amphid and phasmid sensory neurons.

GPCRs as drug targets

* 40% of all prescription drugs on the market target GPCRs.
* Expressed in all tissues, so being investigated for many diseases.
* Cancer, cardiac care, diabetes, obesity, CNS disorders, pain and inflammation.
* Most GPCR drugs were identified on the basis of their activity in a functional assay.
* Not rationally designed based on structure.
* A simple model such as C. *elegans* may be a tool to understand human disease and identify new drugs.