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The Role of PHA-‐4/FoxA Transcrip8on Factor in Establishing the Cellular Context for in vivo forced Transdifferen8a8on
Alexander Atamian
Mentor: Misty Riddle
Faculty advisor: Professor Joel Rothman Molecular, Cellular, and Developmental Biology Department EUREKA, UCSB August 29, 2013
TransdifferenLaLon alternaLve path to treat damaged organs
Regenexx.com
The Model Organism: Caenorhabdi+s elegans
Wormatlas.org
Hermaphrodite Males
How we ask cells to change idenLty in C. elegans
HSP elt-‐7
elt-‐2
Heat Shock
Promoter
IFB-‐2
Normal Expression
gut transcripLon factor
Gut promoter::green fluorescent protein (GFP)
Gut promoter::cyan fluorescent protein (CFP)
The Pharynx and SomaLc Gonad TransdifferenLate into IntesLne; Why?
somaLc gonad pharynx
PHA-‐4 is expressed in the pharynx, intesLne, and somaLc gonad
Wormatlas.org
somaLc gonad
RNA interference of PHA-‐4
double stranded RNA
RNA induced silencing complex
Target mRNA
rnaiweb.com
IFB-‐2 expression is increased in pharynx of PHA-‐4 depleted worms
19 11 11 20 N: 0%
10%
20%
30%
40%
50%
60%
males hermaphrodites
% with
severe CFP in pharynx
Gut marker in Pharynx
Control
pha-‐4(RNAi)
elt-‐2 expression is increased in pharynx of PHA-‐4 depleted worms
0%
20%
40%
60%
80%
posterior pharynx anterior pharynx
% with
GFP in pharynx
Gut marker in Pharynx
Control
pha-‐4(RNAi)
N: 30 30 31 31
Increased number cells with of gut marker in pharynx acer PHA-‐4 depleLon
0
2
4
6
8
10
12
14
1 2 3
# GFP nuclei
Control (N=11) pha-‐4(RNAi) (N=21)
Gut marker in Posterior Pharynx
0
2
4
6
8
10
12
14
1 2 3
# GFP nuclei
Control (N=11) pha-‐4(RNAi) (N=20)
Gut marker in Anterior Pharynx
one C. elegans
Percent worms with IFB-‐2 in somaLc gonads decrease in males and increase in hermaphrodites
19 11 11 20 N: 0%
4%
8%
12%
16%
male hermaphrodite
% with
ectop
ic CFP
Gut marker in Soma8c Gonad
Control
pha-‐4(RNAi)
somaLc gonad
PHA-‐4 is not necessary for ectopic gut differenLaLon in pharynx and uterus
Cyan gut marker in uterus, but not in vas deferens
Increased Cyan and Green gut marker in pharynx of PHA-‐4 depleted worms
-‐Look into other genes responsible for prevenLng transdifferenLaLon of pharyngeal cells
Future Work
Acknowledgements
Mentor: Misty Riddle Advisor: Professor Joel Rothman
Arica Lubin, Kevin Moore, Maria Napoli, Ofelia Aguirre