The Effect of Ectopic Expression of elt-7 on C. elegans’ Chemosensation

By Robert A. SumnerMentor: Misty Riddle

Background

• C. elegans is a model organism– Receptiveness to transgenes and RNAi– Known cell lineage– Known cellular anatomy

http://en.wikipedia.org/wiki/C_elegans

Endoderm cascade genes

http://www.faculty.ucr.edu/~mmaduro/papers/Maduro%20and%20Rothman%20(2002).pdf

During the development of the C. elegans’ endoderm, genes are turned on in a cascade to promote the specialization of the endoderm cells into intestinal cells.

Skin Neurons

Ectopic ELT-7 activates elt-2::LacZ::GFP in neurons, skin, and the pharynx

Pharynx

The elt-7- expressing C. elegans also arrest as L1s.

They stop developing as if they don’t have any food, even if there is food around them

http://www.wormatlas.org

Could their chemosensation be affected?

Agar Plate • Collect hs-end-1 or hs-elt-7 synchronized L1 larva

• Heat shock at 33°C for 15 minutes

• Transfer to middle line• Count number of

worms on each side of the plate after 24 hours

Food

Positive Control: No food on plateControl Experimental

Negative Control: Un-heat-shocked wormsControl Experimental

The number of worms on each hemisphere of the plate was then counted

The number of worms on each hemisphere was divided by the total number of worms on the plate; this number was used to quantify the level of attraction the C. elegans had towards the food on their plate

Ectopic ELT-7 and not END-1 leads to defects in chemosensation

0.25 0.50 0.75 1.00

END-1 No/Yes No

END-1 No Yes

END-1 Yes Yes

ELT-7 No/Yes No

ELT-7 No Yes

ELT-7 Yes Yes

Repulsion No Preference Attraction

Inducing Heat Food Factor -shock

P<0.0001

Ectopic end-3 leads to lesser defects in chemosensation

0.4 0.5 0.6 0.7 0.8 0.9 1

End-1 No NoEnd-1 Yes NoEnd-1 No YesEnd-1 Yes YesEnd-3 No NoEnd-3 Yes NoEnd-3 No YesEnd-3 Yes Yes

Repulsion No Preference Attraction

IF HS Food

P<0.0001

0.5 1

3 Minute Heat-shock has similar effect on chemosensation to No Heat-shock

ELT-7 0min Yes

ELT-7 3Min Yes

ELT-7 15min Yes

Repulsion No Preference Attraction

Inducing Heat Food Factor -shock

0.5 0.6 0.7 0.8 0.9 1

No Significant Difference in Chemosensation After Al-lowing L1 C. elegans to Arrest

No Arrest END-1

No Arrest ELT-7

No Arrest End-3

Arrested END-1

Arrested ELT-7

Arrested End-3

Repulsion No Preference Attraction

Data suggests that the further along the gene expressed is in gut development, the more the

worms’ chemosensation is affected.

end-3: 0.73end-1: 0.98

elt-7: 0.54

Gene: Level of Attraction

Future Directions

• Testing whether the elt-7 expression in the neurons or pharynx is damaging the worms’ chemosensation

• Seeing if a three-minute heat-shock is enough to simply express elt-7 but not reprogram the worms’ pharyngeal cells

• Ectopically expressing elt-7 and other gut developmental genes in different worm stages (L2, L3, L4s, Adults) and seeing how it affects the worms

Acknowledgments

• The Research Mentorship Program• My Mentor: Misty Riddle• Joel Rothman and the Rothman Lab• California Institute for Regenerative

Medicine