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