Jason Manley Drs. Andrea Bodnar & Helena Reinardy 3 October 2014.
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Transcript of Jason Manley Drs. Andrea Bodnar & Helena Reinardy 3 October 2014.
TISSUE REGENERATION Insight from the sea urchin
Jason Manley
Drs. Andrea Bodnar &Helena Reinardy
3 October 2014
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.
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
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!)