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Jason Manley Drs. Andrea Bodnar & Helena Reinardy 3 October 2014.

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1 Jason Manley Drs. Andrea Bodnar & Helena Reinardy 3 October 2014

2 Bermuda Institute of Ocean Sciences

3 Why sea urchins?  More closely related to humans than other invertebrate model organisms  Molecular tools are available for their study  Regeneration of spines & tube feet provide a useful model for muscle, nerve and skin regeneration  Searching for pathways required for tissue regeneration  Understanding regeneration could explain how they maintain tissues over their incredible life span

4 Lytechinus variegatus SEM of sea urchin tube foot

5 Regeneration Assay RNA Extraction & qRT-PCR Goal #1: Investigate the mechanisms of regeneration in L. variegatus using chemical and genetic inhibitors. Goal #2: Measure expression of stem cell marker genes in L. variegatus tube feet.

6 Tube Feet Regeneration Assay

7 Notch signaling has been shown to be important for maintaining cellular proliferation in mice, humans, and during fin regeneration in zebrafish (Moellering, Conboy & Grotek) DAPT: chemical inhibitor of γ-secretase; indirect Notch inhibitor

8 % Regrowth with DAPT Tube FeetSpines Student’s t-test compared to control: * P<0.05 ** P<0.01

9 Vivo-morpholinos: Genetic inhibition  Morpholinos knock down genes: bind to mRNA to prevent translation into proteins  Designed to readily enter cell membranes

10 Regrowth with vMO Tube FeetSpines

11 Tissue Collection RNA Extraction Reverse Transcription qRT-PCR Data: relative levels of gene expression RNA cDNA Amplified DNA

12 High expression Low expression

13 Regeneration, Notch & Stem Cells  Validation of this novel sea urchin regeneration assay  Use of chemical and genetic inhibitors of Notch signaling showed that Notch is critical for regeneration in L. variegatus  Early indication of the presence of stem cells in L. variegatus, when they have never been characterized in echinoderm  Looking forward: advances in human regenerative medicine; understanding how we age (and how we can prevent it!)


15 THANKS ! Drs. Andrea Bodnar & Helena Reinardy

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