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
Lytechinus variegatus SEM of sea urchin tube foot askabiologist.asu.edu/sites/default/files/tubefeetsem.jpg
Regeneration Assay RNA Extraction & qRT-PCR http://bitesizebio.s3.amazonaws.com/wp-content/uploads/2011/11/pipetting-RSI.jpg 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.
http://cdn.zmescience.com/wp-content/uploads/2013/02/jagged1.jpg 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
% Regrowth with DAPT Tube FeetSpines Student’s t-test compared to control: * P<0.05 ** P<0.01
Vivo-morpholinos: Genetic inhibition Morpholinos knock down genes: bind to mRNA to prevent translation into proteins Designed to readily enter cell membranes https://biochemist01.files.wordpress.com/2013/04/central-dogma.jpg
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!)