1. Corbett S. Hall
Harding University, Department of Biology, Searcy, Arkansas, 72149
To address this question, Centanin et al. in 2011 begin experimenting with a Japanese Rice Fish
(Oryzias latipes) model which ubiquitously expressed green fluorescent protein; the model was
named Wimbledon. The researchers transplanted 10-15 Wimbledon+/+ blastula cells into the central
tip of an unlabeled blastula; this region corresponds to the adult retina of the Japanese Rice Fish. At 7
dpf, the fish were screened for GFP-positive retina. Centanin et al. noticed that the retinas developed
arched continuous stripes (ArCoS) in which retinal cells arising from the CMZ migrated posteriorly
over time. These ArCoS could be utilized to help ascertain the potency of a retinal stem cell (RSC)
population. If RSCs are monopotent, the resulting ArCoS should present in only one of the three
retinal layers. However, if RSCs are multipotent, the ArCoS should contribute to all three layers of the
neural retina. The researchers found that an ArCoS arising from one RSC population would develop
into all three major retinal layers. Furthermore, a close look at the ArCoS radial boundary allowed the
scientists to identify multiple cell types based on morphology. Centanin et al. concluded that retinal
stem cells are multipotent during embryonic development.
One of the defining characteristics of life is the ability to grow and develop.
Tissue differentiation is a hallmark of embryonic development, beginning with the
three primary germ layers. Stem cells within the ectoderm germ layer will ultimately
develop into neural or epidermal tissue, and most will lose their potency after the
embryonic phase. However, some neural stem cells are maintained within small niches
and can proliferate throughout life. Within the retina of teleost fish, a conserved stem
cell niche called the ciliary marginal zone has been characterized. The eGFP model
Wimbledon and other fluorescent protein model derivatives were developed to help
assess the developmental potential of retinal stem cells within the CMZ. Fluorescent
protein labeled cells were transfected into the central tip of unlabeled host blastula. As
the labeled cells grew from the ciliary marginal zone towards the optic nerve, arched
continuous stripes were formed. Researchers found that retinal stem cells gave rise to
all retinal nuclear layers and all major cell types. Further, each ArCoS developed from a
single retinal stem cell, confirming the single clonal origin of all retinal cell types.
Retinal stem cells are, therefore, multipotent and maintain their developmental
potential into the post-embryonic stage.
Determining the Developmental Restriction of
Retinal Stem Cells in the Japanese Rice Fish
Abstract
References
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Gene data for this paper were retrieved from the Zebrafish Model Organism Database (ZFIN), University of Oregon, Eugene, OR 97403-5274; URL:
https://zfin.org/ZDB-GENE-040426-2707; 3-22-2016.
One of the defining characteristics of life is the ability to grow and develop. In
vertebrates, including fish and frogs, this process begins with the development of the
three germ layers: endoderm, mesoderm, and ectoderm (Cogle et al., 2003; Menon et
al., 2016). These tissues will develop into the internal organs, connective tissue, and
neural tissue. Within these germ layers, stem cells have varying levels of potential.
Totipotent stem cells can develop into the entire organism, including the placental
tissue; pluripotent stem cells can develop into all major embryonic tissue; multipotent
stem cells can develop into multiple cell types; and monopotent stem cells can only
develop into a single cell type. The developmental process witnesses the exchange of
increased specialization for a decreased potency. Most embryonic stem cells (ESCs)
lose their potency during development; however, select populations can retain this
developmental potential and are maintained inside stem cell niches. Neural stem cell
niches allow for continued neurogenesis in the adult organism. One such example of a
stem cell niche is the ciliary marginal zone (CMZ) (Wehman et al., 2005). This niche is
conserved within frogs and fish, and the retinas of these organisms grow inward from
the CMZ towards the optic nerve.
Stem Cells and Development
Centanin et al. in 2011 generated four multicolor transgenic lines of Japanese Rice Fish: Wimbledon
(expresses nonlocalized eGFP), USOpen (expresses membrane-tagged Cerulean), Aussie (expressed
nuclear-tagged eGFP), and Roland Garros (expressed nuclear-tagged mCherry). The researchers used
a similar transplantation experiment as before to generate adjacent ArCoS with different fluorescent
protein expression. All ArCoS analyzed were composed of cells of a single color. These results confirm
the single clonal origin of arched continuous stripes, which arise from a single RSC population.
Modified “Grand Slam” Transgenic Lines to
Determine Single or Multiclonal Origin
Although the researchers had established the multipotency of RSCs within a developing
organism, more experiments were required to determine if adult animals loss this potential. Centanin
et al. in 2014 developed a Gaudí fluorescent protein reporter line, named after the Spanish mosaic
artist. The system uses a Hsp70.A promoter coupled to a “red-switch-green” Cre/lox recombinase
system. The researchers would heat the fish at 7 dpf, inducing the Hsp70 promoter and changing
fluorescent protein expression from DSRed to eGFP. This approach allowed the researchers to analyze
only post-embryonic retinal development. The team found that post-embryonic RSCs develop into all
three retinal layers and all retinal cell types. This observation suggests that post-embryonic retinal
stem cells retain their multipotency and can develop into all retinal layers of the Japanese Rice Fish.
Addressing Potential Loss of Multipotency with
Developmental Progression
• 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
Conclusions
CMZ
Figure 1 – Methylene-blue staining demonstrates the morphology of the CMZ. Danio rerio larvae were fixed at 6 dpf, horizontal sections were collected
using an ultramicrotome, and samples were stained with 1% azure II-1% methylene blue. No stats were performed. ON = optic nerve, wt = wild-type.
The CMZ is a retinal stem cell niche within teleost. Image taken from (Wehman et al., 2005).
Figure 2 – Wimbledon Model Japanese Rice Fish Allowed for Tracking of ArCoS and Confirmed the Multipotency of RSCs. (a) Wimbledon+/- Cells
Transplanted Into Central Tip of Host Blastula. Wimbledon+/- fish express eGFP ubiquitously during adult stages. 10-15 Wimbledon+/- cells were
transplanted into unlabeled host blastulae, allowing for long-term tracking of these cells and their progeny within the embryonic retina. No stats were
performed. 83% of retinae contained Wimbledon cells, n=136. Wimbledon+/- fish can be transplanted into host blastulae and enable long-term tracking
of retinal stem cells using eGFP expression. (b) An Arched Continous Stripe (ArCoS) demonstrating the migration of RSC-derived neurons towards the
posterior of the retina. (c) Retinal stem cells are multipotent and give rise to all major cell types. Wimbledon+/- cells were transplanted into host
blastulae as previously described. A perpendicular section of the ArCoS revealed that all three nuclear layers were fluorescent. A close-up of the ArCoS
radial boundary facilitated identification of major cell types based on morphology. Imaged using binocular camera. Analyzed 500 ArCoSs in >70 retinae;
“every ArCoS analyzed consisted of a continuous column of EGFP+ cells that spans the three nuclear layers” (556). Retinal stem cells are multipotent and
give rise to all retinal cell types.
(a) (b)
(c)
Figure 3 – Modified Wimbledon Fish Make
Grand Slam Transgenic Models to Confirm
Single Clonal Origin of ArCoS. Researchers
developed a Grand Slam model of transgenic
Japanese Rice Fish. The Wimbledon promoter
was isolated and used to drive expression of
Brainbow 2.1 (USOpen), H2A-Cherry (Roland
Garros), and H2B-EGFP (Aussie) fluorescent
protein systems. 8-15 blastula cells from
USOpen, Aussie, Roland Garros, or
Wimbledon donor fish were transplanted into
an unlabeled blastula. Fish imaged using
binocular camera. Perpendicular sections of
the affected retina revealed ArCoS in close
proximity but of different colors, confirming
that ArCoS originate from a single RSC. No
stats were performed.
n = 76 ArCoSs in 17 retinae.
(a) (b)
Figure 4 – Gaudí Fluorescent Protein Reporter Lines Utilized to Establish Post-embryonic RSC Multipotency. (a) A Hsp70.A promoter was inserted up-
stream of a “red-switch-green” Cre/lox recombinase system. Fish would express DsRed until heat shock, when they switch to expressing H2B-eGFP.
Researchers induced heat shock 7dpf and fixed the retina at 100dpf. (b) Post-embryonic RSCs develop into three major retinal layers. Perpendicular
sections imaged with a binocular camera reveal fluorescent protein expression in all three retina nuclear layers. n > 300; “every single NSC analyzed in
the retina is multipotent” (3476). Post-embryonic RSCs maintain their multipotency.
Acknowledgements
The author would like to thank Dr. Rebekah Rampey for serving as advisor throughout the preparation of this project. In
addition, special thanks to Dr. Mike James, dean of the Honors College, and Ms. Debbie Baird, administrative assistant of
the Honors College; both have been invaluable and supportive during the Honors Capstone process. As always, thanks to
Stephanie Inabnet, who has been a perennial source of encouragement.