Understanding the Molecular Mechanisms of Totipotency using C. - PowerPoint PPT Presentation

Understanding the Molecular Mechanisms of Totipotency using C. elegans By Chijioke Nze (CJ) Mentor: Dr. Nate Dudley Rothman Lab - MCDB

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 Adult animal 1 cell - hatching Totipotent Pluripotent Multipotent Restricted Potential

Totipotency: extension of the competency window Heroes:-Claire-Bear • Identify/Characterize genes required for maintaining totipotency Early development late development T o t i p o t e n t Pluripotent Restricted Potential • Injury and Disease - including tissue loss, many cancers, multiple sclerosis, Alzheimer's etc.

Understanding the role of humpty , in maintaining totipotency Early development Late development Wild type = control Totipotent Pluripotent Multipotent Restricted Potential 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 Early development Late development Wild type = control Totipotent Pluripotent Multipotent Restricted Potential Over-expressed humpty T o t i p o t e n t Pluripotent Restricted Potential

Methods:- Establishing control • Culture of worms • Building the competency window Competency time course 100 80 percent transformed 60 (-) heat shock 40 (+) heat shock 20 0 8-14 cell 12-22 cell 22-42 cell 26-51 cell 46-86 86-170 Approximate cell stage

Competency window assay Candidate dsRNA Heat-Shock END-3 elt-2 GFP HEAT-SHOCK

Heat-shock @ 33 0 C Incubate for 24 hr @ 20 0 C Heat-shock @ 33 0 C Incubate for 24 hr @ 20 0 C

Testing the effects of genetic manipulation • RNAi • Score effects of knocking out humpty on competency • Building a transgenic worm, via microinjection, that over expresses

The competency window appears to be bimodal Percent transformed 100 90 80 94 70 1 47 Percent transformed 60 26 10 33 50 42 34 40 30 45 5 20 10 12 0 1 cell 2-8 cells 10-20 cells22-40 cells42-80 cells ~ 85 cells ~100 cells ~170 cells 190- 300 ventral bean >300 cells cells cleft stage embryo stage

Knocking out humpty has no significant effect within the early period of the window Effect of humpty and Unc-62 RNAi on competency w/ sterror 100 90 80 70 Percent transformed 60 50 Control 40 Unc 62 Nos 2 30 20 10 0 1 cell 2-10 cells 10-20 cells 20-40 cells 40-80 cells ~85 cell ~100 cells ~170 cells 190- 300 ventral bean stage cells cleft Cell Stage

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 genes competency 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 the Rothman lab • Dr. Nate Dudley • UCLEADS • Matt and Arica • CNSI • The audience

Questions??

Heat-shock @ 33 0 C Incubate for 24 hr @ 20 0 C Heat-shock @ 33 0 C Incubate for 24 hr @ 20 0 C

RNAi mechanism