TISSUE REGENERATION Insight from the sea urchin

Jason Manley

Drs. Andrea Bodnar &Helena Reinardy

3 October 2014

Bermuda Institute of Ocean Sciences

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 variegatusSEM 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.

Tube Feet Regeneration Assay

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 inhibitorof γ-secretase;indirect Notch inhibitor

% Regrowth with DAPT

Tube Feet

Spines

Student’s t-testcompared 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

Viv

o-m

orp

holin

o

https://biochemist01.files.wordpress.com/2013/04/central-dogma.jpg

Regrowth with vMO

Tube Feet

Spines

8 15 200%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Days post amputation (dpa)

Regr

owth

(Mea

n &

± S

D)

8 15 20

Control

Delta

SuH

Delta + SuH (n=1)

Days post amputation (dpa)

TissueCollection

RNA Extraction

Reverse Transcription

qRT-PCR

Data: relative levels ofgene expression

RNA

cDNA

Amplified DNA

http://www.thermoscientific.de/com/CMA/Images/Image_44186.jpg

Vasa Oct SoxB1 Myc PCNA Piwi TERT0

1

2

3

4

5

6

7

8

9

10

Stem Cell & Cell Proliferation Markers in Homeostatic Tube Feet

Rela

tive

Gen

e Ex

pres

sion

(ΔCt

)

High expression

Low expression

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!)

THANKS!

Drs. Andrea Bodnar & Helena Reinardy