Upload
others
View
8
Download
0
Embed Size (px)
Citation preview
Understanding the Molecular Mechanisms of Totipotency using C.
elegans
By Chijioke Nze (CJ)Mentor: Dr. Nate Dudley
Rothman Lab - MCDB
https://grid.cnsi.ucsb.edu:9443/gridsphere/images/cnsi_75_white.gif�
C. elegans is a Great Model Organism
• small ~1mm• Short generation time ~ 3 days• Known developmental pattern• Transparent • Genome is sequenced
The C. elegans 1-cell embryo harbors the potential to generate many specialized cell
types
Pluripotent Restricted PotentialMultipotentTotipotent
1 cell - hatching Adult animal
Totipotency: extension of the competency window
• Identify/Characterize genes requiredfor maintaining totipotency
Early development late development
• Injury and Disease - including tissue loss, many cancers, multiple sclerosis, Alzheimer's etc.
Heroes:-Claire-Bear
T o t i p o t e n t Pluripotent Restricted Potential
Understanding the role of humpty, in maintaining totipotency
Wild type = control
Pluripotent Restricted PotentialMultipotentTotipotent
Early development Late development
humpty
Totipotent Multipotent R e s t r i c t e d P o t e n t i a l
Over-expression of humpty
• we hypothesized that by over expressing humpty, we could increase competency
Wild type = control
Over-expressed humpty
T o t i p o t e n t Pluripotent Restricted Potential
Pluripotent Restricted PotentialMultipotentTotipotent
Early development Late development
Methods:- Establishing control• Culture of worms
• Building the competency window
0
20
40
60
80
100
8-14 cell 12-22 cell 22-42 cell 26-51 cell 46-86 86-170
perc
ent t
rans
form
ed
Approximate cell stage
Competency time course
(-) heat shock
(+) heat shock
http://www.google.com/imgres?imgurl=http://www.mrgadget.com.au/catalog/images/rock_remote_control_windows_vista_RM-VR1_0.gif&imgrefurl=http://www.mrgadget.com.au/gadget/2008/rock-remote-control-for-windows-vista-more-power-to-ya/&usg=__dfupRgKmGP4PuguPxKHX98Jjzbo=&h=300&w=300&sz=18&hl=en&start=4&um=1&itbs=1&tbnid=iq43LMwIK-VbbM:&tbnh=116&tbnw=116&prev=/images?q=remote+control&um=1&hl=en&sa=N&tbs=isch:1�
Competency window assay
GFPelt-2
END-3Heat-Shock Candidate dsRNA
HEAT-SHOCK
Heat-shock @ 33 0C
Incubate for 24 hr @ 20 0C
Heat-shock @ 33 0C
Incubate for 24 hr @ 20 0C
Testing the effects of genetic manipulation
• RNAi• Score effects of knocking
out humpty on competency• Building a transgenic worm,
via microinjection, that overexpresses
The competency window appears to be bimodal
12
42
94
26
47
3433
455
0
10
20
30
40
50
60
70
80
90
100
1 cell 2-8 cells 10-20 cells22-40 cells42-80 cells ~ 85 cells ~100 cells ~170 cells 190- 300 cells
ventral cleft
bean stage
>300 cells
Per
cent
tran
sfor
med
embryo stage
Percent transformed
1
10
Knocking out humpty has no significant effect within the early period of the window
0
10
20
30
40
50
60
70
80
90
100
1 cell 2-10 cells 10-20 cells 20-40 cells 40-80 cells ~85 cell ~100 cells ~170 cells 190- 300 cells
ventral cleft
bean stage
Perc
ent t
rans
form
ed
Cell Stage
Effect of humpty and Unc-62 RNAi on competency w/ sterror
Control
Unc 62
Nos 2
Conclusion
• The Competency window of wild-type worms appears to be (stats need to be done to be conclusive) bi-modial with peaks at 20-40 cells and 100
• Humpty does not influence competency early in the window, competency in this period might be regulated by another gene
Future direction
• Define humpty and other related genescompetency early and late in the window
• Create a construct for the over expression of humpty
• Create a worm that over-expresses humpty via micro-injecting of our construct
• Define the competency window for the transgenic worm that over expresses humpty, and score the effects late in development.
Acknowledgements
• Professor Joel Rothman and the amazing people in theRothman lab
• Dr. Nate Dudley• UCLEADS• Matt and Arica• CNSI• The audience
Questions??
Heat-shock @ 33 0C
Incubate for 24 hr @ 20 0C
Heat-shock @ 33 0C
Incubate for 24 hr @ 20 0C
RNAi mechanism
Understanding the Molecular Mechanisms of Totipotency using C. elegans�C. elegans is a Great Model Organism �The C. elegans 1-cell embryo harbors the potential to generate many specialized cell types�Totipotency: extension of the competency window�Understanding the role of humpty, in maintaining totipotencyOver-expression of humptyMethods:- Establishing controlCompetency window assaySlide Number 9Testing the effects of genetic manipulationThe competency window appears to be bimodalKnocking out humpty has no significant effect within the early period of the windowConclusionFuture direction AcknowledgementsSlide Number 16Slide Number 17RNAi mechanism