Understanding the Molecular Mechanisms of Totipotency using C.

elegans

By Chijioke Nze (CJ)Mentor: Dr. Nate Dudley

Rothman Lab - MCDB

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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

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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