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Potency of Neural Stem Cells within the Retina of Japanese Rice Fish
1. Potency of Neural Stem
Cells within the Retina
of Japanese Rice Fish
Corbett Hall
Biology Seminar and Honors Capstone
October 31, 2020
2. Stem Cells and Development
• Ability to grow:
characteristic of life
• Development
requires tissue
differentiation
• Three germ layers:
• Ectoderm
• Mesoderm
• Endoderm
10/31/2020 Corbett Hall Seminar 2
(Cogle et al., 2003; Menon et al., 2016) http://classes.midlandstech.edu/carterp/Courses/bio210/chap04/Slide32.JPG
3. Stem Cells and Development
• Stem cells have the
potential to specialize
• Levels of potency
• Totipotent
• Pluripotent
• Multipotent
• Monopotent
10/31/2020 Corbett Hall Seminar 3
(Cogle et al., 2003; Menon et al., 2016)
Potency
Specialization
Ectoderm Mesoderm Endoderm
Blastocyst
/ Blastula
• Increasing
specialization =
decreasing potency
4. Intestine
Stem Cell Niches
• Limited embryonic
stem cell (ESC)
population remains
potent in adult
• Examples of adult
stem cell niches:
• Hematopoietic (blood)
• Mesenchymal (bone)
• Epithelial (GI tract)
10/31/2020 Corbett Hall Seminar 4
(Doetsch, 2003; Spradling et al., 2001) http://www.nature.com/nrg/journal/v14/n8/images/nrg3471-f2.jpg
Hair follicle
Brain
Hematopoietic system
Skin
Testis
Bone marrow
Skeletal muscle
5. Neural Stem Cell Niches
• Two classic niches within
the mammalian CNS:
• Subventricular zone (SVZ)
• Subgranular zone (SGZ)
• Maintain populations of
neural stem cells (NSCs)
• Allow for continued
neurogenesis in adult
• Less studied niche in frogs
and fish: ciliary marginal
zone
10/31/2020 Corbett Hall Seminar 5
SVZ
(Doetsch, 2003; Lin and Lacovitti, 2015)
SGZ
6. Ciliary Marginal Zone in Teleost
• Frog and fish retina continue
to grow throughout life
• Ciliary marginal zone (CMZ)
is conserved niche within
frogs and fish
• Retina grows inward from
CMZ, towards the optic nerve
(Wehman et al., 2005)
6
CMZ
10/31/2020 Corbett Hall Seminar
7. Ciliary Marginal Zone in Teleost
• Teleost (“complete skeleton”)
fish models include:
• Japanese Rice Fish (Oryzias latipes)
• Zebrafish (Danio rerio)
• Retinal stem cells (RSCs)
within the CMZ develop into
two distinct tissues:
• Neural retina (NR)
• Retinal-pigmented epithelium (RPE)
(Centanin et al., 2011; Harris and Perron, 1998; Wehman et al., 2005)
710/31/2020 Corbett Hall Seminar
http://faculty.washington.edu/tomreh/images/eyedev2.jpg
Embryo
Adult
RPE
RPE
NR
NR
9. Question 1:
What is the developmental
restriction of retinal stem cells in
the Japanese Rice Fish?
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10. Wimbledon+/- Cells Transplanted Into
Host Blastula
10/31/2020 Corbett Hall Seminar 10
(Rembold et al., 2006; Centanin et al., 2011)
10-15
Wimbledon+/+
blastula cells
Wild-type
http://clipart-finder.com/data/mini/31-blastula.png
Screened
for GFP
positive
retina
11. RSCs Generate Arched Continuous
Stripes during Development
• Labeled RSCs develop
towards optic nerve
• Form Arched
Continuous Stripes
(ArCoS)
• Youngest neurons
near the CMZ, oldest
near the embryonic
retinal line
10/31/2020 Corbett Hall Seminar 11
(Centanin et al., 2011)
Time
Post-embryonic
retina
CMZ
Lens
ArCoS
embryonic
retina
12. 1210/31/2020 Corbett Hall Seminar
Expected Histology of Cell Type-
Specific and Multipotent RSCs
(Centanin et al., 2011)
• GCL = Ganglion
Cell Layer
• INL = Inner
Nuclear Layer
• ONL = Outer
Nuclear Layer
13. 1310/31/2020 Corbett Hall Seminar
RSCs Develop into Three Major
Retinal Layers
(Centanin et al., 2011)
14. 1410/31/2020 Corbett Hall Seminar
(Centanin et al., 2011)
Modified Wimbledon Fish to Make
Grand Slam of Transplanted Models
Roland Garros
15. 10/31/2020 Corbett Hall Seminar 15
(Rembold et al., 2006; Centanin et al., 2011)
8-10 Grand
Slam blastula
cells
Wild-type
http://clipart-finder.com/data/mini/31-blastula.png
Modified Wimbledon Fish to Make
Grand Slam of Transplanted Models
16. 1610/31/2020 Corbett Hall Seminar
(Centanin et al., 2011)
Adjacent yet Different ArCoS Reveal
Single Clonal Origin
17. Conclusion 1:
Embryonic retinal stem cells in
Japanese Rice Fish are:
•Multipotent
•The clonal origin of arched
continuous stripes
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18. Question 2:
How does developmental
progression affect the
multipotency of retinal stem cells
in the Japanese Rice Fish?
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19. Gaudí Fluorescent Protein Reporter
Lines Driven by Hsp70 Promoter
10/31/2020 Corbett Hall Seminar 19
(Centanin et al., 2014; Livet et al., 2007) http://www.biography.com/people/antoni-gaudí-40695; http://whc.unesco.org/en/list/320
20. Cre/lox Recombination Inducible
by Heat Shock in GaudíRSG
10/31/2020 Corbett Hall Seminar 20
(Centanin et al., 2014; Livet et al., 2007)
Hsp70
promoter
Cre
recombinase
lox
lox
lox
lox
Inversion
21. Gaudí Fluorescent Protein Reporter
Lines Driven by Hsp70 Promoter
10/31/2020 Corbett Hall Seminar 21
(Centanin et al., 2014)
Induction of Clones
Fix
100dpfday 0
HS!
7dpf
HS!
Hsp70
promoter
Cre
recombinase
lox
lox
lox
lox
22. Post-embryonic RSCs Develop into
Three Major Retinal Layers
10/31/2020 Corbett Hall Seminar 22
(Centanin et al., 2014)
23. Conclusion 2:
Post-embryonic retinal stem cells
retain their multipotency and
develop into all major retinal
layers in the Japanese Rice Fish
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25. Final Conclusions
• Retinal stem cells are multipotent and
develop into all three neural retina layers
• The neural retina has a single clonal
origin; each ArCoS comes from one RSC
• Developmental progression does not
affect the multipotency of RSCs
• Japanese Rice Fish retina provide an
ideal model for vertebrate retina
development
10/31/2020 Corbett Hall Seminar 25
27. Stem Cells and Development
• Limited embryonic
stem cell (ESC)
population remains
potent in adult
• Reside in stem cell
niches which
provide:
• Somatic cell
signaling
• Basement membrane
• Extracellullar matrix
10/31/2020 Corbett Hall Seminar 28
(Doetsch, 2003; Spradling et al., 2001)
28. Dentate Gyrus of Hippocampus
as Stem Cell Niche
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http://www.cell.com/pictureshow/brainbow
29. Ciliary Marginal Zone in Teleost
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http://faculty.washington.edu/tomreh/images/eyedev2.jpg
Time in Development
30. Wimbledon+/+ Medaka Contain GFP
Cassette in hnrnpl Gene
10/31/2020 Corbett Hall Seminar 31
(Centanin et al., 2011; ZFIN: hnrnpl)
heterogeneous nuclear ribonucleoprotein L2
GFP
31. Cre/lox Recombination Inducible
by Heat Shock in GaudíLxBBW
10/31/2020 Corbett Hall Seminar 32
(Centanin et al., 2014; Livet et al., 2007)
Hsp70
promoter
Cre
recombinase
lox
lox
lox
lox
lox
OR
lox
lox
Exclusions
lox
lox
lox
Inversions
32. Palette of Fluorescent Proteins
10/31/2020 Corbett Hall Seminar 33
(Tsien, Nobel Lecture)
Editor's Notes
Thank you Dr. Rampey
As she mentioned, my name is CH, and today I will be discussing the Potency of Neural Stem Cells within the Retina of Japanese Rice Fish
The ability to grow is a defining characteristic of life.
Development process requires tissue differentiation
In vertebrates, including fish and frogs, process starts with development of three germ layers: endo-, meso-, ecto- which develop into…
We will focus on the ectoderm layer today.
Stem cells within these germ layers have the potential to specialize.
However, various levels of potential: toti through mono-
As specialization increases, potency decreases.
Focusing today on blastocyst ESCs ectoderm neural tissue
Most ESCs lose their potency in adulthood
However, some retain potency.
Examples: hematopoietic (blood cells), mesenchymal (bone cells & adipose), epithelial (GI tract)
Typically, no neuroregeneration in adult CNS
Two well-studied niches: subventricular zone, subgranular zone
Both maintain NSCs in mammalian brain
Allow for continued neurogenesis; BIG DEAL!
We need to study these niches more… what about the CMZ?
However, retinal stem cell niche has not been well understood...
Niche we discuss today lies within the retina of frog and fish, which grow throughout life
CMZ is conserved, and retina grows outward towards ON
Wehmen et al. 2005 Semi-thin plastic sections demonstrate the cellular morphology of the CMZ in mutants. (A) Methylene-blue-stained horizontal sections of the 6 dpf larval retina. (A′) Magnifications of the boxed areas from A. The CMZ is between the paired arrowheads. (A and A′) Wild-type. ON: Optic nerve. Scale bar in A–D, 50 μm. Scale bar 10 μm.
Focus on Teleost (“complete skeleton”) fish models, b/c link to vertebrates…
JRF (Oryzias latipes) and Zebrafish (Danio rerio)
Two requirements: (1) reliable method to produce clones and (2) tools for long-term lineage analysis
Model for vertebrate retinal development
RSCs within CMZ NR and RPE (mention abbreviations)
Explain figure (from embryo to adult, CMZ is preserved as niche!)
Three layers of the neural retina: outer nuclear layer (cones and rods), inner nuclear layer (bipolar cells), ganglion cell layer (retinal ganglion cells)
Studies will investigate whether all three layers arise from one RSC
My question…
Centenan et al. 2011
ubiquitously EGFP-expressing fish that we named Wimbledon
Transplanted 10-15 Wimbledon+/− blastula cells to the central tip of an unlabeled WT JRF blastula
Focus on retina 7 dpf, retinal cross-section here
RSCs develop towards optic nerve from ciliary marginal zone (spell out)
Form ArCoS (animation)
Newest neural cells near CMZ, oldest near embryonic retinal line
Point out three nuclear layers
If RCSs are cell type specific, they should form ArCoS containing just a single cell type (center); on the contrary, multipotent RSCs will generate ArCoS that contain all different cell types (right).
GCL – ganglion cell layer, INL – inner nuclear layer, ONL – outer nuclear layer
RSCs Are Multipotent
(B) A section perpendicular to the ArCoS reveals that the all cell types across the three nuclear layers are fluorescent.
(C) A closer look at the radial boundary of the ArCoS facilitates the identification of the different cell types based on their exclusive morphology.
GCL – ganglion cell layer, INL – inner nuclear layer, ONL – outer nuclear layer
MORE TIME
(1) Wimbledon, expressing nonlocalized EGFP and DS-Red, (2) USOpen, expressing a membrane-tagged Cerulean, (3) Aussie, expressing a nuclear-tagged EGFP, and (4) Roland Garros, expressing a nuclear-tagged Cherry,
Multicolor Transplantations Confirm the Clonal Origin of the ArCoSs
(A) A Grand Slam of four trangenic medaka expressing different fluorescent proteins with distinct subcellular localization were used as donors in cotransplantation experiments.
(B) ArCoSs formed by a combination of cell-specific RSCs should display different cell types in different colors (left). On the contrary, if ArCoSs are clonal all of them should be composed of just one color (right).
Centenan et al. 2011
ubiquitously EGFP-expressing fish that we named Wimbledon
Transplanted 10-15 Wimbledon+/− blastula cells to the central tip of an unlabeled WT JRF blastula
Focus on retina 7 dpf, retinal cross-section here
Multicolor Transplantations Confirm the Clonal Origin of the ArCoSs
On left, Roland Garros and Aussie
On right, Wimbledon and USOpen
(C–E) All ArCoS analyzed were composed of cells of a unique color, even ArCoSs in close proximity (C and E), demonstrating their clonal origin.
Scale bars represent 50 μm.
Embryonic retinal stem cells in Japanese Rice Fish are:
Multipotent
The clonal origin of arched continuous stripes
How does developmental progression affect the multipotency of retinal stem cells in the Japanese Rice Fish?
Centanin et al. 2014 made Gaudi fluorescent protein model Japanese Rice Fish, named after Spanish artist known for mosaics.
Mosaic cells in retina.
Used Hsp70.A promoter, so inducible by heat shock
Various reporter genes, based on Brainbow from Livet et al., 2007 that uses Cre/lox recombinase system
Gaudi RSG = Gaudí Red-Switch-Green
More time spent here!
HS! 7 dpf, fixed 100 dpf
See results on right different fluorescent expression induced post-embryonic via HS!
More time here, walk through each slide - SLOW
Cross sections of retina using Hsp70, LxBBW reporter
Showed expression in all three nuclear layers
Reminder of cell type/nuclear layer correlation – explain, emphasize different cell types
Post-embryonic retinal stem cells retain their multipotency and develop into all major retinal layers in the Japanese Rice Fish
Most ESCs lose their potency in adulthood
However, some retain potency.
Stem cell niches stabilize these adult ESCs by providing them with... Somatic cell signaling, basement membrane, ECM
