Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 821
Characterization of Retinal
Progenitor Cells
Focus on Proliferation and the GABAA Receptor
System
HENRIK RING
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2012
ISSN 1651-6206
ISBN 978-91-554-8489-7
urn:nbn:se:uu:diva-180011
Dissertation presented at Uppsala University to be publicly examined in B41, BMC,
Husargatan 3, Uppsala, Thursday, December 13, 2012 at 10:00 for the degree of Doctor of
Philosophy (Faculty of Medicine). The examination will be conducted in English.
Abstract
Ring, H. 2012. Characterization of Retinal Progenitor Cells: Focus on Proliferation and
the GABAA Receptor System. Acta Universitatis Upsaliensis. Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Medicine 821. 60 pp. Uppsala.
ISBN 978-91-554-8489-7.
One strategy to repair an injured or degenerated retina is to stimulate the replacement of damaged
or dead neurons with cells derived from endogenous stem- or progenitor cells. A successful
strategy requires knowledge about how the proliferation and differentiation of the endogenous
cells are regulated. In particular, this knowledge will be important in the establishment of
protocols that produce sufficient numbers of specific neurons. The main aim of this thesis was to
find and characterise factors regulating the proliferation and differentiation of retinal progenitor
cells (RPCs) and hence, contribute to the knowledge of how to use progenitor cells for retinal
repair.
The major result in this thesis is that GABA contributes to and maintains RPC proliferation.
Inhibition of GABAA receptors decreases the proliferation of non-pigmented ciliary epithelial
(NPE) cells and RPCs in the intact retina. We propose that this effect is mediated through
changes in the membrane potential and voltage-gated calcium channels, which in turn regulate
components of the cell cycle. Furthermore, we show that one of the endogenous RPC sources,
the Müller cells, consists of two subpopulations based on Pax2 expression. This is interesting
because Pax2 may suppress the neurogenic potential, characterised by de-differentiation
and proliferation, in Müller cells. Finally, we show that over-expression of FoxN4 induces
differentiation-associated transcription factors in the developing chick retina. However, FoxN4
over-expression did not trigger differentiation of NPE cells. These results indicate that the
intrinsic properties of the RPCs are determinant for FoxN4-induced differentiation.
The results presented in this thesis advance our understanding of how specific cells may be
generated from different sources of RPCs. Our results show that the different sources are highly
diverse in their potential to proliferate and produce neurons. GABA, Pax2 and FoxN4 may be
factors to consider when designing strategies for retinal repair. However, the results indicate
that the specific responses to these factors are highly associated with the specific properties of
the progenitor cells.
Keywords: Regeneration, Proliferation, Neurotransmitters, Müller cells, Differentiation,
Retinal repair, Neurogenesis
Henrik Ring, Uppsala University, Department of Neuroscience, Developmental Neuroscience,
Box 587, SE-751 23 Uppsala, Sweden.
© Henrik Ring 2012
ISSN 1651-6206
ISBN 978-91-554-8489-7
urn:nbn:se:uu:diva-180011 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-180011)
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Boije, H., Ring, H., Lopez-gallardo, M., Prada, C. & Hallböök, F.
(2010) Pax2 is expressed in a subpopulation of Müller cells in the
central chick retina. Dev Dyn 239, 1858-1866.
II Ring, H.*, Boije, H.*, Daniel, C., Ohlson, J., Öhman, M. &
Hallböök, F. (2010) Increased A-to-I RNA editing of the transcript
for GABAA receptor subunit alpha3 during chick retinal
development. Vis Neurosci, 27, 149-157.
III Ring, H., Mendu, SK., Shirazi-Fard, S., Birnir, B. & Hallböök, F.
(2012) GABA maintains the proliferation of progenitors in the
developing chick ciliary marginal zone and non-pigmented ciliary
epithelium. PLoS ONE 7(5): e36874.
IV Boije, H., Shirazi-Fard, S., Ring, H. & Hallböök F. (2012) Forkhead
box N4 (FoxN4) triggers context-dependent differentiation in the
developing chick retina and neural tube. (Submitted to
Differentiation)
*equal contribution
Reprints were made with permission from the respective publishers.
Contents
Introduction ................................................................................................... 11
The retina.................................................................................................. 11
The structure and cells of the retina ..................................................... 11
Development of the eye ....................................................................... 12
The cell cycle and control of cell proliferation ........................................ 13
Mitogenic signalling pathways ............................................................ 15
The Notch signal transduction pathway............................................... 17
Effects of neurotransmitters on cell proliferation ................................ 17
Neurogenesis in the developing retina ................................................. 19
Retinal regeneration ................................................................................. 21
Ciliary marginal zone .......................................................................... 22
Retinal pigment epithelium.................................................................. 23
Müller cells .......................................................................................... 23
Ciliary epithelium ................................................................................ 24
The GABAA receptor system ................................................................... 25
GABAA receptor subunits .................................................................... 27
GABAA receptor agonists/antagonists ................................................. 28
GABA expression and effects in the retina ......................................... 28
Aims of the thesis.......................................................................................... 29
Results and Discussion ................................................................................. 30
Paper I ...................................................................................................... 30
Paper II ..................................................................................................... 32
Paper III .................................................................................................... 35
Paper IV ................................................................................................... 37
Conclusions ................................................................................................... 40
Material and Methods ................................................................................... 43
Animals .................................................................................................... 43
Immunohistochemistry ............................................................................. 43
Quantitative reverse transcription PCR .................................................... 43
Proliferation analyses: EdU assay, [3H]-thymidine incorporation and
MTT assay ................................................................................................ 44
Flow cytometry ........................................................................................ 45
Electrophysiology..................................................................................... 46
In situ hybridization ................................................................................. 46
Sequencing ............................................................................................... 47
Electroporation ......................................................................................... 48
Acknowledgements ....................................................................................... 49
References ..................................................................................................... 51
Abbreviations
α3
AC
ANOVA
bFGF
BP
CDK
CDKI
CMZ
CNS
E(number)
EGF
FACS
FoxN4
GABA
GCL
HC
IHC
INL
IPL
KCC2
MC
NPE
ONL
OPL
Pax2
Pax6
PR
Rb
GC
RPC
RPE
SR-95531
St(number)
TGF
VGCC
Alpha3
Amacrine cell
Analysis of variation
Basic fibroblast growth factor
Bipolar cell
Cyclin-dependent kinase
CDK inhibitor
Ciliary marginal zone
Central nervous system
Embryonic day
Epidermal growth factor
Fluorescence-activated cell sorting
Forkhead box N4
Gamma-amino butyric acid
Ganglion cell layer
Horizontal cell
Immunohistochemistry
Inner nuclear layer
Inner plexiform layer
K+-Cl- co-transporter
Müller glia cell
Non-pigmented ciliary epithelium
Outer nuclear layer
Outer plexiform layer
Paired box gene 2
Paired box gene 6
Photoreceptor cell
Retinoblastoma gene
(Retinal) ganglion cell
Retinal progenitor cell
Retinal pigment epithelium
Gabazine
Embryonic stage, according to
(Hamburger & Hamilton 1951)
Transforming growth factor
voltage-gated calcium channel
Preface
Vision is an amazing sense most seeing people more or less takes for
granted. It is however easy to imagine the complex processes that are
required for creating visual perceptions.
About 40 million people in the world are blind. Approximately 35% of
these cases of blindness are caused by retinal diseases or injuries (Pascolini
& Mariotti 2012). The human retina has probably a limited ability for
endogenous repair, which has resulted in comprehensive research with focus
on replacing damaged or degenerated retinal neurons through
transplantations. Photoreceptor neurons can be transplanted into an adult
retina where they integrate with the host retinal neurons and thereby restore
damaged or lost functions to a certain degree (Lamba et al 2009). However,
the most successful transplantations have included photoreceptors from late
stage developed foetuses, which raise both ethical and practical issues
(Lamba et al 2009). One way to solve this is through the use of endogenous
stem- or progenitor cells. Interestingly, the human eye was recently reported
to contain quiescent cells that proliferate and develop into photoreceptor-like
neurons in response to retinal injury (Ducournau et al 2011, Johnsen et al
2012). Thus, some endogenous cells in the eye have the potential to be
stimulated and used in order to repair a degenerated or damaged retina.
This thesis focuses on two aspects of retinal repair: cell proliferation and
differentiation. Regeneration including stem- or progenitor cells covers at
least three steps (Gilbert 2010): 1. proliferation of stem- or progenitor cells,
resulting in a pool of cells that can generate specific cells, 2. differentiation
of stem- or progenitor cells, resulting in specific cells that can replace
damaged or dead cells, and 3. migration and integration of differentiated
stem- or progenitor cells, resulting in a restored function. The main focus of
the thesis was on the proliferation of retinal progenitor cells. Further clues of
what regulates the proliferation can widen our knowledge on how to trigger
and maintain proliferation of quiescent endogenous cells. These cells can
then be stimulated to produce sufficient numbers of new neurons.
Introduction
The retina
The retina (Fig. 1) is the part of the central nervous system (CNS) where
vision begins by the transformation of photons (light) to electrochemical
signals, which are further processed in the brain. The retina is composed of
six types of neurons; cone- and rod photoreceptor cells (PRs), horizontal
cells (HCs), bipolar cells (BPs), amacrine cells (ACs) and ganglion cells
(GCs), and one type of glial cell; Müller cells (MCs). The neurons interact
with each other in order to process the incoming visual signals before they
arrive to the brain. The visual stimuli can be either enhanced or silenced
depending on the strength of the visual input, and on how various neurons
respond to and integrate the stimuli.
The structure and cells of the retina
The different cell types in the retina are organized in distinct laminas that are
divided into nuclear and plexiform layers. The cell somas are located in the
nuclear layers and the axons and dendrites in the plexiform layers. The
layers are termed the outer nuclear layer (ONL), the outer plexiform layer
(OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), the
ganglion cell layer (GCL) and the optic fibre layer (OFL). The ONL contains
the cone- and rod PRs, which are responsible for converting light into
electrical signals. Cone PRs are active during bright light conditions, for
example during normal daylight, and initiates high-resolution perceptions
that discriminate visual details. There are three types of cone PRs in the
retina. Each cone PR has a specific photo pigment that can absorb light of a
specific wavelength, which in turn makes the cone PRs able to detect
colours. Rod PRs, on the other hand, are extremely sensitive to light and are
used during dim light conditions (Kaufman & Alm 2003). The INL contains
the cell bodies of MC, HCs, BPs and ACs. HCs are a class of interneurons
that are involved in enhancing contrast between light and dark regions. They
act by integrating and modulating the output from PRs to BPs by means of
inhibitory synapses. BPs act as relay stations for visual signals, which means
that they relay information from PRs to the GCs. ACs integrate and
modulate the signals from BPs to GCs by both excitatory and inhibitory
synapses. MCs are the primary glial cell of the retina and they support the
11
retinal structure and regulate retinal homeostasis. The GCL contains GCs
and displaced ACs. GCs are the last neurons in the retina to receive the
visual signals and their function is to convey the information to the brain.
Figure 1. The organization of the vertebrate retina. AC, amacrine cell; BP, bipolar
cell; GC, ganglion cell; GCL, ganglion cell layer; HC, horizontal cell; INL, inner
nuclear layer; IPL, inner plexiform layer; MC, Müller cell; OFL, optic fibre layer;
ONL, outer nuclear layer; OPL, outer plexiform layer; PR, photoreceptor cell (rods
and cones); RPE, retinal pigment epithelium.
Development of the eye
Eye development begins as a single eye field located in the anterior neural
plate. The development continues as the neural plate invaginates to form the
neural tube in the midline of the embryo, in a process called neurulation.
During the establishment of the midline of the embryo, the single eye field is
separated and during the neurulation, the first signs of developing eyes
appear as two lateral optic pits. Within a few days these optic pits develop
into optic vesicles (Fig 2A), which are connected to the developing forebrain
(derived from the neural tube) by the optic stalks (giving rise to the later
optic nerve). When the optic vesicles contact the surface ectoderm, inductive
events take place to cause the ectoderm to form lens placodes. The lens
12
placodes will later invaginate, pinch off and eventually become the lenses.
Once the formation of the lens placodes has begun, the optic vesicles begin
to invaginate and fold along their centrelines in order to form the bilayered
optic cups (Fig 2B). The outer layer of the optic cups will become the retinal
pigment epithelium and the inner layer the neural retina (Fig. 2C) (Gilbert
2010, Graw 2010).
Figure 2. Development of the eye. See corresponding text for details. ov, optic
vesicle; RPE, retinal pigment epithelium.
Multipotent retinal progenitor cells (RPCs) are present in the central part of
the inner layer of the optic cups and have the ability to generate all the cell
types of the retina (Marquardt & Gruss 2002). The generation of the retinal
cells is often divided into an “early phase” when GCs, HCs and cone PRs are
born, and a “late phase”, when BPs, rod PRs and MCs are born. The ACs are
generated in between these phases (Prada et al 1991b, Reese 2011). Most of
the retinal cells in the human eye are produced between the 10th and 19th
week of gestation (Martins & Pearson 2008). Cell generation in the retina
will be further discussed in the chapter “Neurogenesis in the developing
retina” of this thesis.
The cell cycle and control of cell proliferation
During early development, the rate of proliferation is high in most
prospective organs (Malumbres & Barbacid 2001). In adult animals
13
however, cell proliferation is low and limited to tissues in which new cells
are needed to maintain organ size or for other functions (Lodish 2008,
Malumbres & Barbacid 2001, Morgan 2007). The rate of proliferation is
regulated by a combination of intracellular and extracellular factors. The
intracellular factors depend on tissue-specific genetic programming and
extracellular factors are produced by other cells.
Proliferating cells participate in the cell cycle (Fig. 3), which usually is
divided into four phases: G1, before DNA synthesis occurs; S, when DNA
replication occurs; G2, after DNA synthesis; and M, when cell division
occurs, resulting in two daughter cells. If a cell has left the cell cycle it is in a
phase called G0 (Lodish 2008). The rate of proliferation is usually regulated
at a cell-cycle checkpoint in late G1 phase, sometimes called the “restriction
checkpoint” (Fig. 3) (Morgan 2007). Proteins that stimulate proliferation,
usually referred to as mitogens (Morgan 2007), and other regulatory factors
influence the proliferation rate by controlling the components that regulate
progression through the G1 checkpoint. These components are known as
cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKI)
(Abukhdeir & Park 2008, Sherr & Roberts 1999). The CDKs are active only
when they bind to their specific cyclins in order to form complexes that
trigger progression through different stages of the cell cycle by
phosphorylating specific target proteins (Abukhdeir & Park 2008,
Malumbres & Barbacid 2001).
Progression through the G1 checkpoint depends on the expression of G1/S
genes, which is controlled by regulatory proteins such as members of the
E2F transcription factor family (Malumbres & Barbacid 2001, Morgan
2007). The E2F transcription factors are regulated primarily by the
retinoblastoma (Rb) proteins (Malumbres & Barbacid 2001). In quiescent
cells, E2F is bound to Rb proteins, which suppresses the expression of E2F
target genes and inhibits cell cycle progression. Mitogens promote entry to
the cell cycle by activating cyclin-CDK complexes, usually cyclin D and its
partners CDK4 and CDK6. The complexes phosphorylate Rb proteins
resulting in the release of the E2F transcription factors, which in turn enable
expression of G1/S genes (Abukhdeir & Park 2008, Sherr & Roberts 1999).
14
Figure 3. The cell cycle and its most important cyclin-CDK complexes. The cell
cycle is regulated by cyclins and cyclin-dependent kinases (CDKs), which form
complexes that trigger advancement in the cycle by phosphorylating specific
proteins. The cyclin D-CDK4/6 complex phosphorylates the retinoblastoma (Rb)
protein, which releases the regulatory protein E2F. E2F then promotes expression of
other cyclin-CDK complexes and thereby induces cell cycle progression.
The assembly of active cyclin D-CDK complexes requires cofactors such as
the CIP/KIP family proteins p27KIP1 and p21CIP1/WAF1, which activate cyclin
D-CDK4 but inhibit the cyclin E-CDK2 and cyclin A-CDK2 complexes
(Abukhdeir & Park 2008, Sherr & Roberts 1999). The cyclin E/A-CDK2
complexes are required for initiation of DNA replication (Abukhdeir & Park
2008) and the removal of the CDK-inhibitors is therefore crucial for cell
cycle progression.
Mitogenic signalling pathways
Mitogens, such as basic fibroblast growth factor (bFGF) and epidermal
growth factor (EGF), are well-known stimulators of the proliferation of
progenitors in the retina (Close et al 2005, Fischer & Bongini 2010, Fischer
& Reh 2002, Fischer & Reh 2003, Pittack et al 1991). In principle, mitogens
stimulate proliferation by binding to specific cell surface receptors, usually
receptor tyrosine kinases (RTKs). The binding causes dimerization of two
receptor molecules, which activates protein kinase domains present on the
intracellular region of the receptor. The activated kinases phosphorylate
tyrosine residues, which create new binding sites on the receptor. The new
sites can bind intracellular signalling proteins, for instance proteins
containing the SH2 family of protein domains. The Ras pathway (Fig. 4) is a
common pathway that mitogens activate. When mitogens activate their
15
receptor, specific phosphorylated tyrosine residues interact with an SH2containing protein called Grb2. Grb2 binds to a protein called Sos, which is a
guanine-nucleotide exchange factor for a small GTPase called Ras. Ras is
activated in the presence of GTP but turns inactive when GTP is hydrolysed
to GDP. In order to re-activate Ras Sos removes GDP, which enables new
GTP to bind Ras.
Figure 4. The Ras/MAPK cascade and Notch signal transduction pathways. (a) In
the Ras/MAPK cascade, a mitogen activates its receptor, usually a receptor tyrosine
kinase (RTK). The intracellular domain of the receptor becomes phosphorylated and
interacts with an adapter protein called Grb2. Grb2 binds to a cytosolic protein
called Sos. Sos activates Ras (a GTPase), which in turn activates the Raf kinase. Raf
activation triggers the MAPK cascade, which will result in a dimeric form of active
MAP kinase. The MAP kinase dimer translocates into the nucleus and activates
transcription factors (TF) in order to stimulate transcription of genes (e.g. c-Fos). (b)
In the Notch signal transduction pathway, binding of a ligand to the Notch receptor
triggers proteolytic cleavage of the receptor, which releases the intracellular domain.
The released intracellular domain translocates to the nucleus where it activates
transcription factors.
Ras promotes cell proliferation mainly through the MAP kinase cascade. In
the MAP kinase cascade a kinase called Raf is activated by binding to
activated Ras. Raf activates another kinase, MEK, which then activates the
MAP kinase. The activated MAP kinase leads the mitogenic signal to the
nucleus where it phosphorylates, and thereby activates, several regulatory
16
proteins. These proteins induce the expression of immediate early genes, for
instance c-Myc and c-Fos. c-Fos forms a dimer with a member of the c-Jun
family of gene regulatory proteins. The dimer is known as the Activator
Protein 1 (AP-1), which inactivates the CDKI p27KIP1 (Khattar & Kumar
2010, Yang et al 2001). The inactivation of p27KIP1 promotes expression of
cyclins and thereby leads to cell cycle progression (Adhikary & Eilers 2005,
Sherr & Roberts 1999) (Fig. 10).
The Notch signal transduction pathway
The Notch signal transduction pathway (Fig. 4) is involved in deciding
whether a cell should continue to proliferate or leave the cell cycle in order
to differentiate. Notch is a transmembrane receptor protein that is interacting
with both extracellular and intracellular proteins. The extracellular activation
of the Notch receptor is mediated by the ligands Delta or Jagged (Ghai et al
2010). Activation of the Notch receptor induces a proteolytic cleavage and a
release of the intracellular Notch domain. The released intracellular domain
activates expression of the Hairy and enhancer of split homolog (Hes) genes
(Ghai et al 2010). Neural stem cells are kept in a proliferative state when the
Hes genes are expressed since these repress the transcription of the proneural genes Mash and Neurogenin 2 (Castella et al 1999, Holmberg et al
2008). Thus, Notch signalling represses pro-neural genes and promotes cells
to be kept as progenitor- or glial cells (Gaiano et al 2000, Jadhav et al 2006).
Neuronal differentiation can be promoted by inhibiting Notch signalling by
for example the γ-secretase inhibitor DAPT (Nelson et al 2006, Nelson et al
2007).
Effects of neurotransmitters on cell proliferation
Classical neurotransmitters such as glutamate and gamma-amino butyric
acid (GABA) have been ascribed to other features beside their roles as
transmitters of information between neurons. Over the last two decades there
have been several studies establishing the involvement of both slow and fast
acting neurotransmitters in the regulation of cell proliferation. All of the
common neurotransmitters such as acetylcholine, glycine, dopamine,
serotonin, glutamate and GABA have been found to regulate the
proliferation of a variety of cell types both within and outside the CNS
(Andäng et al 2008, Berg et al 2011, Fiszman et al 1999, Kralj-Hans et al
2006, Luk et al 2003, Ma et al 2000, Martins et al 2006, Ohtani et al 2003,
Pearson et al 2002, Schwirtlich et al 2010, Toriumi et al 2012, Wang &
Kriegstein 2009, Vitalis & Parnavelas 2003, Young & Cepko 2004). Many
of the neurotransmitters can both stimulate and inhibit cell proliferation
depending on the cell type (Table 1). For example, glutamate decreases the
proliferation of neuronal progenitors of the cerebral cortex (Toriumi et al
17
2012) but increases the proliferation of progenitors of the striatum (Luk et al
2003). Another example is GABA that decreases the proliferation of
embryonic stem (ES) cells (Andäng et al 2008, Schwirtlich et al 2010) and
neuronal stem- and progenitor cells (Fernando et al 2011, Wang &
Kriegstein 2009) but promotes proliferation of cerebellar progenitor cells
(Fiszman et al 1999). Neurotransmitters may therefore act through different
cellular pathways depending on the intrinsic properties of the cell type. For
example, GABA depolarises neuronal progenitor cells and immature
neurons, which results in an efflux of chloride ions and an influx of calcium
ions (Ca2+) (Ben-Ari 2002, Ben-Ari et al 2007, Eilers et al 2001, Yamashita
& Fukuda 1993, Yuste & Katz 1991). Increased concentration of
intracellular Ca2+ can activate the MAPK cascade pathway (Hilgenberg &
Smith 2004), which in turn has been shown to both stimulate and suppress
cell proliferation (Fiszman et al 1999, Schwirtlich et al 2010).
The ability to modulate concentrations of intracellular Ca2+ is a feature
common to all the early neurotransmitter systems and often plays an
important role in the regulation of cell proliferation (Gu & Spitzer 1995)
(Lee et al 2006, Whitaker & Larman 2001).
In Paper III of this thesis, we analysed the effects of the GABAA receptor
system on the proliferation of non-pigmented ciliary epithelial- (NPE) and
retinal progenitor cells. We also examined through what mechanism the
system acts.
18
Table 1. Effects of some neurotransmitters on cell proliferation
Effect on cell
Neurotransmitter
Cell type (species)
proliferation
cortical progenitors
stimulation
Acetylcholine
(rat)
retinal progenitors
inhibition
(chicken)
retinal progenitors
inhibition
Glycine
(mouse)
Dopamine
Serotonin
Glutamate
GABA
cortical progenitors
(mouse)
stimulation and
inhibition
retinal progenitors
(rat)
inhibition
neural stem cells
(newt)
cortical progenitors
(rat)
inhibition
stimulation
hippocampal cells
stimulation
cortical progenitors
(rat/mouse)
inhibition
retinal progenitors
(mouse)
inhibition
embryonic stem cells
(mouse)
inhibition
immune cells (rat)
inhibition
neural stem cells
(mouse)
inhibition
cortical and retinal
neurons (chicken)
stimulation
Reference
(Ma et al 2000)
(Pearson et al
2002)
(Young & Cepko
2004)
(Ohtani et al
2003, Popolo et
al 2004)
(Kralj-Hans et al
2006, Tibber et al
2006)
(Berg et al 2011)
(Vitalis &
Parnavelas 2003)
(Arnold & Hagg
2012)
(Haydar et al
2000, LoTurco et
al 1995)
(Martins et al
2006)
(Andäng et al
2008, Schwirtlich
et al 2010)
(Mendu et al
2011)
(Fernando et al
2011, Liu et al
2005)
(Spoerri 1988)
Neurogenesis in the developing retina
The multipotent RPCs that generate the neurons and glial cell of the retina
are heterogeneous and give rise to clones of different sizes and cell type
compositions in culture (Holt et al 1988, Turner et al 1990). The
transcription factor Ikaros, a homolog of Drosophila hunchback (Pearson &
Doe 2004), has been suggested to specify competence for early cell fates.
Ikaros protein is expressed in early but not in late mouse RPCs, and is
required to generate the early-born cell types (Elliott et al 2008). This
intrinsic regulation could explain why RPCs differentiate independently of
19
the environment in which they develop and why young RPCs cannot be
induced to generate older cell types and vice versa (Cepko et al 1996,
Livesey & Cepko 2001). Another important transcription factor expressed in
early retinal progenitors is Sox2. Sox2 has been shown to stimulate both
proliferation and differentiation of neuronal progenitor cells. It promotes
proliferation and suppresses neuronal differentiation in the chick spinal cord
(Bylund et al 2003, Graham et al 2003). However, Sox2 triggers neuronal
fate in the mouse retina. A genetic ablation of Sox2 pushes RPCs towards a
ciliary epithelium (CE) fate in which they stay undifferentiated (Matsushima
et al 2011, Taranova et al 2006). A mechanism has been suggested where the
transcription factors Sox2 and Pax6 compete with each other; high
expression of Sox2 decreases the expression of Pax6 and bias RPCs towards
a neurogenic fate, whereas high expression level of Pax6 decreases the
expression of Sox2 and bias the RPCs towards a CE fate (Matsushima et al
2011).
Lateral inhibition, governed by the Notch signal transduction pathway
(Fig. 4), is an important mechanism during retinal development in deciding
if a RPC should stay as an undifferentiated cell or leave the cell cycle in
order to differentiate. RPCs that express Notch ligand (for example Delta)
and have high proneural activity differentiate into neurons whereas cells
with high Notch activity remain as progenitors or generate MCs (Hayes et al
2007).
GCs are the first and MCs are the last retinal cells to be generated. The
differentiation of the retina follows a central to peripheral gradient. This
means that early-born cell types in the peripheral retina are generated after
late-born cell types in the central retina (Agathocleous & Harris 2009). The
differentiation gradient is regulated by FGF signalling that promotes
expression of the transcription factor Ath5 (also known as Atoh7 or Math5),
which is necessary for differentiation of GCs (Martinez-Morales et al 2005,
McCabe et al 2006, Wang et al 2001, Willardsen et al 2009). The initiation
of GC differentiation leads to a domino effect where newly differentiated
cells signal to their neighbours to leave the cell cycle and differentiate. Sonic
hedgehog (Shh) signalling, initiated by the ligand Shh secreted from GCs
(Amato et al 2004, Wang et al 2005), has been ascribed to be crucial for
RPCs to leave the cell cycle in order to differentiate (Shkumatava &
Neumann 2005). Shh signalling has also been shown to induce expression of
cyclinD1 and Hes1 in developing mouse retinas resulting in increased cell
proliferation (Wang et al 2005). This was however shown to be accompanied
with a decreased multipotency of the RPCs (Cwinn et al 2011).
Transcription factors including members of the basic helic-loop-helix
(bHLH), homeodomain and forkhead families have been ascribed as key
intrinsic regulators of retinal cell fate. Some examples are Otx2 and Crx that
are required for differentiation of PRs (Chen et al 1997, Nishida et al 2003);
and Ptf1a, Prox1, Sall3 and FoxN4 for HCs (de Melo et al 2011, Dyer et al
20
2003, Fujitani et al 2006, Li et al 2004). The role of FoxN4 during retinal
development was further examined in Paper IV of this thesis.
Retinal regeneration
Already in the 18th century, scientists showed that adult newts have the
ability to regenerate the retina after injury. In the end of the 19th century, the
Italian-Swiss scientist Colucci (reviewed in Keefe, 1973) proposed that cells
at the peripheral retinal margin (the ciliary marginal zone; CMZ) was the
primary source of cells behind the regeneration (Keefe 1973, Leigh Close &
Reh 2006). This was confirmed in later studies, which also demonstrated that
the retinal pigment epithelium (RPE) could regenerate the newt retina (Leigh
Close & Reh 2006).
Until quite recently the general opinion has been that nothing can be
regenerated in the mammalian retina (Zupanc & Sirbulescu 2011). However,
even though the mammalian ability of endogenous repair or regeneration is
still considered to be very low compared to that in fish or amphibians, there
is a growing body of evidence of areas in the mammalian eye that are
analogues to adult stem- or progenitor cell niches found in non-mammalian
vertebrates (Fig. 5) (Wohl et al 2012).
In this thesis, four different sources of cells with potential to repair or
regenerate the retina will be discussed: CMZ, RPE, MCs and Ciliary
epithelium (CE). The different cellular sources for repair or regeneration
share several common features: 1. they are all derived from the neural tube,
2. they all have the capacity to re-enter the cell cycle, and 3. they all express
several genes expressed in retinal progenitors (Leigh Close & Reh 2006,
Wohl et al 2012).
21
Figure 5. Localisation of sources of cells with the potential to contribute to retinal
regeneration in the mammalian (human) eye. CMZ, ciliary marginal zone; MC,
Müller cell; NPE, non-pigmented ciliary epithelium; RPE, retinal pigment
epithelium.
Ciliary marginal zone
The ciliary marginal zone (CMZ), also known as the ora serrata or
circumferential germinal zone (Fig. 5) (Bhatia et al 2011), is situated
between the neural retina and the ciliary body. CMZ is a source of new
retinal neurons in many amphibians and fish (Raymond et al 2006, Wohl et
al 2012, Zupanc & Sirbulescu 2011). The CMZ in teleost fish retina contains
multipotent stem cells, which give rise to GCs, ACs, BPs, HCs and cone PRs
throughout the life as the eye grows larger (Raymond et al 2006, Zupanc &
Sirbulescu 2011). In vertebrates where most of the development of the retina
is ready before birth, most of the proliferation is decreased after the
embryonic or neonatal period. However, proliferation often remains in the
CMZ. In the chicken, for example, at least 90% of the retinal cells are
generated more than one week before hatching (Prada et al 1991a) but the
CMZ continues to be proliferative in the post-hatched chicken up to one
month of age (Fischer & Reh 2000). The CMZ cells express a number of
different genes that also are expressed in RPCs, for example Pax6, Chx10
(Fischer & Reh 2000) and Notch1 (Fischer 2005). The neurogenic potential
for the avian CMZ cells has however been shown to be fairly poor compared
to fish and amphibians, and chicken CMZ cells normally produce only ACs
and BPs (Fischer 2005). Thus, progenitors in the CMZ may be limited in the
capacity to generate particular neuronal cell types. The proliferation and
neurogenic ability can be stimulated by exogenous growth factors. EGF can
increase the proliferation as much as 10-fold (Fischer & Reh 2000) and a
combination of insulin and bFGF stimulates the production of GCs in
22
addition to ACs and BPs (Fischer et al 2002a). In contrast to fish and
amphibians the post-hatch avian CMZ progenitors are not able to repair or
regenerate the retina after injury (Moshiri et al 2004).
Mammals hold a CMZ-like area with RPCs that have some neurogenic
potential (Bhatia et al 2009, Moshiri et al 2004). The proliferative capacity
of rodent CMZ is rapidly decreased during the first postnatal period but
injury or disease have been shown to trigger CMZ cell proliferation (Jian et
al 2009, Moshiri et al 2004). The adult human eye has also been reported to
contain quiescent neural progenitors in the peripheral retina (ora serrata)
and in the ciliary epithelium of the ciliary body. These progenitors
proliferate and express neural stem cell markers such as Pax6, Sox2 and
Nestin, in response to injury or disease (Bhatia et al 2011, Johnsen et al
2012).
Retinal pigment epithelium
The retinal pigment epithelium (RPE) absorbs stray light and is responsible
for phagocytosis of the outer segments of rod PRs (Wohl et al 2012).
RPE is a cellular source for retinal regeneration in the adult newt
(Beddaoui et al 2012, Nakamura & Chiba 2007). Removal of the amphibian
retina leads to a transdifferentiation of the RPE (Okada 1980). The
transdifferentiation includes a de-pigmentation of the RPE cells, which then
proliferate and generate two new epithelial layers: a pigmented layer and a
non-pigmented layer. The non-pigmented layer expresses genes that are
expressed in RPCs and undergoes extensive cell proliferation to produce
sufficient numbers of neurons for an entire new retina (Reh & Nagy 1987).
The embryonic chick retina is capable of a similar form of RPE
transdifferentiation; if an E3-E4 chick retina is removed, the RPE undergoes
a transdifferentiation into neural retinal progenitors (Park & Hollenberg
1989, Park & Hollenberg 1991). Unlike the newt RPE, the
transdifferentiation of embryonic chicken RPE requires that some part of the
retina still is intact. If the entire retina is removed the RPE will not
transdifferentiate (Coulombre & Coulombre 1965, Park & Hollenberg 1993).
Müller cells
The Müller cells (MCs) are the major type of support cell in the retina
(Fischer & Bongini 2010). They provide structural support, synaptic support,
osmotic homeostasis and metabolic support to retinal neurons (Fischer &
Bongini 2010). MCs and late-born retinal neurons are derived from a
common precursor (Turner & Cepko 1987), and MCs have been shown to
maintain some of the characteristics of neuronal progenitor cells. They have
the capacity in many vertebrates to undergo cell division, express
23
multipotent progenitor cell markers and generate some retinal neurons
(Fischer & Bongini 2010).
In the post-hatch chicken retina insulin and bFGF, or the neurotoxin Nmethyl-D-aspartate (NMDA) stimulate MCs to generate RPC-like cells and
re-enter the cell cycle (Fischer et al 2002b, Fischer & Reh 2001). The
induced de-differentiation and proliferation of MCs involve activation of the
MAPK cascade (Fig. 4) (Fischer et al 2009a, Fischer et al 2009b). The
majority of the newly generated cells remain as undifferentiated progenitorlike cells, while some differentiate into MCs, ACs or BPs (Fischer & Reh
2001). MCs have also been shown to be a cellular source of retinal
regeneration in the zebrafish (Bernardos et al 2007, Zupanc & Sirbulescu
2011) and in the mouse (Karl et al 2008). The retina of teleost fish and
amphibians continues to grow throughout life. The retina grows by addition
of new neurons mainly from the CMZ (Zupanc & Sirbulescu 2011), except
for rod progenitors that are derived from MCs (Bernardos et al 2007). In
mouse and rat retinas, the MCs have a limited ability to proliferate after
severe retinal degeneration (Dyer & Cepko 2000, Moshiri et al 2004). One
reason for the difference between mammals and non-mammalian vertebrates
could be expression of TGF-β2. TGF-β2 is produced by postnatal retinal
neurons and has been shown to inhibit proliferation of MCs (Close et al
2005).
Human MCs in culture proliferate and express neural stem- and
progenitor cell markers (Bhatia et al 2011). Sustained culturing of the cells
transform them into progenitor-like cells that express markers for GCs and
PRs (Bhatia et al 2011). However, evidence for in vivo repair or regeneration
performed by MCs in the primate retina is currently lacking.
In Paper I of this thesis, we investigated possible differences in the
capacity to de-differentiate and proliferate in response to retinal injury in two
subpopulations of Müller cells (MCs): Pax2 expressing (positive) and Pax2
negative MCs.
Ciliary epithelium
Many regions of the anterior eye, for example the ciliary body and iris, have
been proposed to be sources of retinal stem cells (Ahmad et al 2000,
Tropepe et al 2000).
The ciliary epithelium (CE) of the ciliary body (Fig. 5) produces
components of the aqueous humor and does not normally contain neurons
(Fischer & Reh 2003). It is formed from the anterior part of the optic cup
(Venters et al 2011) and has a two-layered structure: a pigmented layer and a
non-pigmented layer (NPE) (Fischer & Reh 2003). The CE can further be
subdivided into the more proximal pars plana and the distal, folded pars
plicata (Fischer & Reh 2003). The NPE cells express the retinal stem cell
transcription factors Pax6 and Chx10 (Ahmad et al 2000, Fischer & Reh
24
2003, Tropepe et al 2000). Chicken NPE cells can be stimulated by growth
factors to generate neurons, but these neurons do not migrate into the neural
retina (Fischer & Reh 2003). Thus, the NPE cells are not a cellular source of
endogenous retinal regeneration. Instead, they have the potential to be
stimulated to generate neurons in culture and then transplanted into injured
retinas in order to replace degenerated neurons (Fischer & Bongini 2010,
Fischer & Reh 2003).
Dissociated cells from the mammalian (mouse) CE in culture undergo
transdifferentiation and give rise to non-pigmented neurospheres (Ahmad et
al 2000, Tropepe et al 2000). These neurospheres express retinal antigens in
the presence of serum (Tropepe et al 2000). Furthermore, they have been
shown to generate PR-like cells in response to specific transcription factors
such as Otx2 and Crx (Akagi et al 2004b, Coles et al 2004, Tropepe et al
2000). These characteristics have given the mouse CE cells the epithet as
being retinal stem cells with potential to generate PRs. This description has
both been challenged (Cicero et al 2009, Gualdoni et al 2010) and confirmed
(Ballios et al 2012, Demontis et al 2012). The opposing studies showed that
mouse CE cells in culture proliferate into non-pigmented neurospheres,
which express retinal stem cell marker genes. However, the neurospheres
were shown to be unable to differentiate into specific retinal neurons (Cicero
et al 2009, Gualdoni et al 2010). This observation has been disproved by
other studies (Ballios et al 2012, Demontis et al 2012). Cultured CE cells
were shown to express rhodopsin (Rho) and other proteins involved in the
phototransduction cascade (Demontis et al 2012). Furthermore, the Rhoexpressing cells could respond to cGMP, a secondary messenger in the
phototransduction cascade, in a similar manner as rods do. These results
indicate that adult mouse CE cells are able to generate functional rod-like
cells (Demontis et al 2012).
In Paper III of this thesis, we used chick NPE cells to analyse if the
neurotransmitter GABA is involved in the proliferation of retinal
progenitors.
The GABAA receptor system
GABA, the main inhibitory neurotransmitter in the adult brain, is one of the
first neurotransmitters to become active during development (Ben-Ari 2002).
Many embryonic cells including neuronal progenitors have high
concentration of intracellular chloride ions (Cl–). Opening of the GABAA
receptor Cl– channels will therefore lead to Cl– efflux and depolarisation of
the cell membrane (Zhao et al 2011). The shift from excitatory to inhibitory
actions by the GABA system is a result of altered Cl– transport and is
regulated by the ubiquitous Na–K–Cl co-transporter (NKCC1) and the
neuron-specific K–Cl co-transporter (KCC2). Increased expression of the
25
KCC2 transporter during CNS development has been described as a key
event in the shift (Ben-Ari 2002, Rivera et al 1999). In Paper II and III of
this thesis, we studied the relative messenger RNA (mRNA) levels of the cotransporters during chick retinal development (Paper II) and in chick NPE
cells (Paper III) in order to determine what effect the GABAA receptor
system had on the cell membrane potential. Furthermore, in Paper III we
analysed if the depolarising currents mediated by GABA is involved in the
proliferation of retinal progenitors. GABA has been implied to regulate
stem- and progenitor cell proliferation (Andäng et al 2008, LoTurco et al
1995), neuronal migration (Behar et al 1996), and activity dependent trophic
interactions (Represa & Ben-Ari 2005, Wang & Kriegstein 2009). These
different actions by one single molecule may be explained by a high degree
of complexity in the constitution of the GABA receptors. GABAA receptors
are the main mediators of fast inhibitory neurotransmission in the mature
mammalian CNS. These receptors are heteropentamers formed from at least
19 different subunits with minor variations among species. In the chicken,
subunits α1-6, β1-4, γ1-3, δ, ε, π and ρ1-3 have been identified (Ring et al
2010). Individual neurons express several subunits resulting in the formation
of structurally and functionally different receptor subtypes. Most GABAA
receptors are composed of two α, two β, and either one γ or δ subunit
(Hevers & Luddens 1998) (Fig. 6).
The GABAA receptors can be subdivided into synaptic and extrasynaptic
receptors (Belelli et al 2009). Inhibition can occur via the conventional
transient activation of GABAA receptors (phasic inhibition), or via
continuous activation of high affinity receptors by low concentrations of
ambient GABA leading to tonic inhibition (Jin et al 2011).
Figure 6. Structure of the GABAA receptor. The GABAA receptors are most
commonly composed of two α, two β, and either one γ or δ subunit. There are two
active GABA binding sites between the α and β subunits.
26
GABAA receptor subunits
The binding site for GABA is located between the α- and β subunits of the
GABAA receptor (Smith & Olsen 1995). Variations in the sensitivity to
GABA has been ascribed to the α subunit of the receptor (Böhme et al 2004,
Ebert et al 1994). Receptors containing the α6 subunit have the highest
sensitivity to GABA whereas α3 subunit containing receptors have the
lowest sensitivity (Böhme et al 2004, Ducic et al 1995, Picton & Fisher
2007).
Synaptic GABAA receptors usually contain the α1, α2, α3 (WinskySommerer 2009) and γ2 subunits (Essrich et al 1998). Tonic (extrasynaptic)
GABAA receptors, located outside of the immediate synapse, often contain
the α4, α5, α6 (Caraiscos et al 2004, Farrant & Nusser 2005, Semyanov et al
2004) and sometimes δ subunits (Fritschy & Brunig 2003, Nusser et al
1998). Activation of GABAA receptors containing the α4 and γ1 subunits
have been shown to decrease the proliferation of cortical precursor cells
(LoTurco et al 1995, Nguyen et al 2003) and neural crest stem cells (Andäng
et al 2008, Represa & Ben-Ari 2005).
The ρ subunits compose the so-called GABAA-ρ receptors that previously
were known as the GABAC receptors. Like other GABAA receptors they are
expressed in many areas of the CNS but in contrast to other GABAA
receptors, the GABAA-ρ receptors have especially high expression in the
retina (Qian & Ripps 2009).
We analysed the mRNA expressions of the GABAA receptor subunits in
order to investigate the presence and constitution of receptors in the
developing chick retina (Paper II) and in the chick NPE (Paper III). We
found that most subunits were expressed above background levels except for
the β1 (retina and NPE cells), π (retina) and α5 (NPE cells).
27
GABAA receptor agonists/antagonists
A number of ligands have been found to bind various sites on the GABAA
receptor and modulate it besides GABA itself.
•
Muscimol is a powerful selective GABAA receptor agonist (Rang
et al 2003).
•
Bicuculline is a specific competitive GABAA receptor antagonist.
Bicuculline reduces GABAA receptor currents by decreasing the
open frequency and mean duration (Macdonald & Olsen 1994).
•
Picrotoxin acts by blocking the chloride channel associated with
the GABAA receptor. It is a non-competitive antagonist (Rang et
al 2003) and closes GABAA receptor channels that are
spontaneously open (Macdonald & Olsen 1994).
•
Benzodiazepines have powerful sedative and anxiolytic effects.
They bind with high affinity to an accessory site on the GABAA
receptor in such way that the binding of GABA is facilitated and
its agonist effect is enhanced.
•
SR-95531 (also known as gabazine) is a selective competitive
GABAA receptor antagonist. It also acts as a low affinity glycine
receptor antagonist (Yeung et al 2003).
GABA expression and effects in the retina
Glutamate is the major neurotransmitter in the retina and mediates the
“direct” visual signal pathway: PR to BP to GC. GABA and glycine on the
other hand mediate the “indirect” pathway, involving HCs and ACs. The
indirect pathway contributes to the complex receptive field properties of
some retinal neurons (Sun & Crossland 2000). GABA is present in several
AC subpopulations and in some cells in the GCL in all vertebrate species
that have been examined (Sun & Crossland 2000). In the avian retina,
GABA is present in most of the HCs, numerous AC subpopulations, and in
some cells in the GCL (Edqvist et al 2008, Sun & Crossland 2000).
28
Aims of the thesis
The main aim of this thesis was to find and characterise different factors
involved in the proliferation and differentiation of retinal progenitor cells in
order to better understand how to use progenitor cells for retinal repair. It is
hoped that the results will provide new insight on how to expand small
endogenous progenitor cell pools, as well as how to trigger progenitors to
develop into specific cell types.
The specific aims were:
Paper I: To investigate possible differences in the capacity to de-differentiate
and proliferate in response to retinal injury in two subpopulations of Müller
cells (MCs): Pax2 expressing (positive) and Pax2 negative MCs.
Paper II: To determine if the transcript of the GABAA receptor α3 subunit is
expressed in the developing chick retina and to analyse if the RNA editing of
the α3 transcript changes during development. We were also interested to
know if a change in the RNA editing was temporally related to the
developmental depolarising-to-hyperpolarising shift of the GABAA system.
Paper III: To examine the effects of the GABAA receptor system on the
proliferation of non-pigmented ciliary epithelial- (NPE) and retinal
progenitor cells. We also aimed to determine through what mechanism the
system acts.
Paper IV: To analyse the role of the transcription factor FoxN4 in chicken
retinal progenitor cells and to determine if FoxN4 is sufficient to commit
cells to a HC fate in the chicken retina.
29
Results and Discussion
Paper I
We discovered that the transcription factor Pax2, a member of the paired
homeobox family, was expressed by Sox2 expressing MCs in the chicken
retina (Fig. 7). Birthdate analysis showed that the majority of Pax2
expressing MCs were born between E5.5 and E8 (Fig. 2A-B in Paper I).
However, the Pax2 expression was restricted to centrally located MCs (Fig.
3A in Paper I). Pax2 defines the ventral region of the optic cup (Fig 1C in
Paper IV) and plays important roles in differentiation processes. Pax2
inhibits neurogenesis in the retina and triggers formation of the optic nerve
and generation of astrocytes. Our hypothesis was that the centrally located
Pax2 expressing MCs were less prone to de-differentiate and proliferate in
response to retinal injury, compared to the Pax2 negative MCs.
In order to study the Pax2 expression among proliferating MCs we
injected growth factors or a toxic component, N-methyl-D-aspartate
(NMDA), together with the S-phase marker EdU, into the eyes of newly
hatched chickens. The growth factor injections induced proliferation of Sox2
positive, Pax2 negative MCs located in the peripheral part of the retina (Fig.
5A-C in Paper I). The NMDA treatment triggered both central and
peripheral MCs to proliferate. These proliferating cells were not expressing
Pax2. There were however, EdU negative MCs in the central part of retina
that expressed Pax2 (Fig 5E in Paper I).
We conclude that Pax2 is a novel marker of a centrally located population
of MCs in the chicken retina. To our knowledge, this is the first time that
MCs have been shown to be a heterogeneous cell population based on gene
expression. Our hypothesis was false; the Pax2 positive MCs in the central
retina were able to de-differentiate and proliferate as a response to retinal
injury. The central EdU positive MCs did not express Pax2 but Sox2 (Fig.
5E in Paper I). Sox2 has been shown to trigger cells to develop into neurons
whereas Pax2 inhibits neuronal fates and promotes glial cell fates. We
therefore suggest that Pax2 is down regulated in cells that go through Sphase in response to injury in order to promote neuronal cell fates. We
further suggest that Pax2 is involved in the differentiation of MCs and that
Pax2 could be important for mature MCs since the expression of Pax2
remains in the adult retina.
30
In parallel to our study, Stanke and colleagues (Stanke et al 2010) also
found that Pax2 is expressed by centrally located MCs. Their results were
somewhat different compared to ours. They found that the expression level
of Pax2 was increased in MCs in response to acute retinal damage or
treatment with growth factors. However, they did not find any Pax2
expression in MCs in peripheral regions of the retina (Stanke et al 2010),
where the MCs are known to be more plastic, proliferative and neurogenic
compared to MCs in central regions of the retina (Fischer 2005). Therefore,
they suggest that Pax2 may suppress the de-differentiation of MCs or
promote glial phenotype in central regions of the retina (Stanke et al 2010).
Recently published results from a study of Pax2 expression during injuryinduced regeneration in the goldfish optic nerve head, support our
conclusion. The expression of Pax2 was not changed compared to control
during the first two days after an induced cryolesion, but from day 7 to 30
after the lesion the expression increased. This suggests that Pax2 may be
involved in late regeneration-stages, for example in differentiation of
progenitor-like cells (Parrilla et al 2012).
31
Figure 7. Central and peripheral expression of Pax2 (red) and Sox2 (green) in the
adult chicken retina. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer
nuclear layer.
Paper II
GABA is the main inhibitory neurotransmitter in the adult CNS. However,
initially it depolarises neuronal progenitors and immature neurons in many
developing networks (Ben-Ari 2002). The shift from excitatory to inhibitory
actions by the GABA system has been ascribed to increased expression of
the KCC2 co-transporter (Ben-Ari 2002, Rivera et al 1999), see the chapter
“The GABAA receptor system” of this thesis.
This study was made in collaboration with Marie Öhman’s group at
Stockholm’s university. The Öhman group previously found that the
transcript for the α3 subunit of the GABAA receptor was subjected to
Adenosine-to-Inosine (A-to-I) RNA editing (Ohlson et al 2007). A-to-I RNA
32
editing is a co- or posttranscriptional process on double-stranded RNA,
catalysed by adenosine deaminases that act on RNA (ADARs). Editing
results in an alteration of the pre-mRNA sequence encoded from the
genome. In A-to-I RNA editing, adenosine is deaminated to inosine, which
is read as guanosine by the translation machinery (Fig. 8A). The editing
results in an amino acid isoleucine-to-methionine (I-to-M) change (Fig. 8B).
Our preliminary data indicated that the transcript for the α3 subunit was Ato-I RNA edited in the adult retina. The aim of this study was to determine if
the transcript for the α3 subunit is expressed in the developing chick retina,
and to analyse if the editing of the transcript changes during development.
We were also interested to know if a change in editing was temporally
related to the developmental depolarising-to-hyperpolarising shift of the
GABAA receptor system, as has been suggested by previous studies (Ohlson
et al 2007, Rula et al 2008). Furthermore, we investigated the localisation of
the α3 subunit mRNA in the retina and the temporal expression of mRNA
for 16 GABAA receptor subunits in the developing chick retina.
Figure 8. A-to-I RNA editing. (A) Adenosine is converted to inosine by adenosine
deaminases that act on RNA (ADARs). Inosine is read as guanosine by the
translation machinery. (B) The A-to-I editing of the GABAA receptor subunit α3
leads to an amino acid isoleucine-to-methionine change.
Our study showed that the transcript for the α3 subunit was subjected to
RNA editing in the chicken retina and brain (Fig 1B-C in Paper II) and that
mRNA expression of the α3 subunit was restricted to cells in the inner
nuclear- and ganglion cell layers of the retina (Fig. 2C-I in Paper II). The
level of A-to-I editing increased during development (after st35/E8-9; Fig.
1D in Paper II) concomitantly with an increase of mRNA expression of the
chloride transporter KCC2 (Fig 9). KCC2 mediates efflux of chloride ions
33
and thereby contributes to the inhibitory effect that GABA has in the mature
CNS. The mRNA expression levels of several GABAA receptor subunits,
known to mediate synaptic GABA actions, were also increased at this time
(Fig. 3 in Paper II).
RNA editing is a mechanism of post-transcriptional control similar to
alternative pre-mRNA splicing, and like alternative splicing, editing plays an
important role in the nervous system by increasing the diversity of the
proteome. Well-characterised substrates for RNA editing are the transcripts
for the glutamate receptors (Rosenthal & Seeburg 2012, Seeburg 2002).
Editing of the GluA2 (AMPA) receptor transcript at the Q/R
(glutamine/arginine) site is essential to the organism and required for a
normal brain development (Brusa et al 1995, Feldmeyer et al 1999). Editing
of the transcript for the GABAA receptor subunit α3 in mice has been shown
to alter the GABA sensitivity and to generate receptors with slower
activation and faster deactivation rates (Nimmich et al 2009, Rula et al
2008). The unedited α3 subunit confers a higher sensitivity to GABA, and
this subunit is the dominating form early when GABA is depolarising (Rula
et al 2008). Therefore, chicken retinal cells that have receptors containing
the unedited α3 subunit may contribute to and allow long-lasting excitatory
responses.
We conclude that RNA editing of the α3 subunit transcript occurs in the
chicken retina and that the extent of the editing increases during the
development. The editing may be of importance during the developmental
depolarising-to-hyperpolarising shift by reducing the GABA sensitivity of
the GABAA receptors.
Figure 9. Relative mRNA levels of the chloride ion co-transporters NKCC1 and
KCC2 during chick retinal development. Line graph shows the relative mRNA
levels of NKCC1 and KCC2 in the developing chick retina from st25 to 45 analysed
by using qRT-PCR. All levels were related to the levels of st45 NKCC1. Error bars
± S.D., n = 4, one-way ANOVA, Tukey’s multiple comparison test ***P < 0.001. st,
Hamburger & Hamilton developmental stage.
34
Paper III
GABA has been implied to regulate stem- and progenitor cell proliferation
(Andäng et al 2008, LoTurco et al 1995, Schwirtlich et al 2010), neuronal
migration (Behar et al 1996) and activity dependent trophic interactions
(Represa & Ben-Ari 2005, Wang & Kriegstein 2009). These different
actions by one single molecule may be explained by a high degree of
complexity in the constitution of the GABAA receptors. In the study
presented in Paper II, we discovered that the expression levels of most
GABAA receptor subunits were increased from st35 (E8-9). The mRNA
level of the co-transporter KCC2 was also increased from this stage. These
findings indicate that GABA causes depolarisation of RPCs at least until E89. The depolarising currents mediated by GABA have been shown to be
important for a number of developmental processes, including proliferation
(Ben-Ari et al 2007). The results from Paper II encouraged us to analyse if
GABA has effects on the proliferation of RPCs. Freshly dissected NPE cells
were used to monitor the effects of GABA because of their homogeneity and
their similarities to early RPCs, such as the expression of Pax6 and Chx10
(Fischer & Reh 2003). The NPE cells expressed 17 of total 19 GABAA
receptor subunits (Fig. 1A in Paper III). The subunits formed functional
GABAA receptors that were activated by low concentrations of GABA (Fig.
1B-C in Paper III). The cells expressed low levels of the chloride transporter
KCC2 (Fig. 1D in Paper III), which suggest that the concentration of
intracellular chloride was relatively high compare to the extracellular levels.
This in turn suggests that the GABAA receptors cause a depolarisation of the
cell membrane when stimulated. The GABAA receptor agonist muscimol did
not have any additional effects on the proliferation of the cells compared to 1
µM GABA while inhibition of the receptors by GABAA receptor antagonists
decreased the proliferation (Fig. 2A in Paper III). The effects on proliferation
by the GABAA receptor antagonists could be counteracted by addition of
extracellular KCl (Fig. 2B in Paper III), a treatment that depolarises the
plasma membrane (Purves 2012). Cells in the intact E8 retina were also
treated with receptor antagonists and this resulted in a decreased
proliferation as well (Fig. 3B-G in Paper III). Embryonic GABAA receptor
systems increase the concentration of intracellular Ca2+ through activation of
voltage-gated calcium channels (VGCCs) (Eilers et al 2001, Yamashita &
Fukuda 1993). Elevated concentration of Ca2+ has been shown to activate
calcium/calmodulin-dependent kinase II (CaMKII) and MAPK signalling
pathways, which in turn activate the immediate early gene complex activator
protein 1 (AP-1) (Hilgenberg & Smith 2004, Morgan & Curran 1988, Zwick
et al 1999). AP-1 suppresses the expression of the CDK inhibitor p27KIP1,
which stimulates the cell cycle to progress (Khattar & Kumar 2010, Morgan
& Curran 1988) (see the chapter “The cell cycle and control of cell
proliferation” in the introduction of this thesis). The proliferation of NPE
35
cells decreased when the cells were treated with the VGCC blocker
nifedipine (Fig. 2B in Paper III). Furthermore, the expression of p27KIP1
increased when the cells were treated with nifedipine or GABAA receptor
antagonists (Fig. 5A, C in Paper III).
The conclusion of the study is that NPE cells and some specific RPCs in
the intact E8 retina express functional GABAA receptors that participate in
the regulation of their proliferation. We suggest that the GABAA receptors
contribute to maintaining the proliferation by regulating the plasma
membrane potential. We propose a mechanism in which activation of
embryonic GABAA receptors causes a depolarisation of the cell membrane,
which in turn activates VGCCs resulting in an increased concentration of
intracellular Ca2+. The Ca2+ causes expression of the immediate early genes
c-Fos and c-Jun, which in turn inhibit the expression of p27KIP1. The
inhibited expression of the CDK inhibitor enables the cell cycle to progress
(Fig. 10).
As a follow-up experiment of the study, we investigated if the
phosphoinositide 3-kinase (PI3K) signalling pathway supported a possible
GABA-mediated Ras/MAPK pathway activation. NPE cells were
electroporated with a vector containing the sequence for green fluorescent
protein (GFP) fused to that of the general receptor for phosphoinositides-1
(Grp1).
The
Grp1
protein
specifically
binds
phosphatidylinositol(3,4,5)trisphosphate (PIP3) (Venkateswarlu et al 1998).
When
the
PI3K
pathway
is
activated,
the
phosphatidylinositol(4,5)bisphosphate (PIP2) is converted to PIP3 through
PI3K. PIP3 recruits protein effectors to the cell membrane resulting in a colocalisation of enzymes and substrates that stimulates further downstream
signalling cascades (Guillou et al 2007). PIP3 also recruits the GFP-Grp1
probe to the membrane. GFP fluorescence intensity at the plasma membrane
is therefore proportional to the PIP3 concentration and consequently reflects
PI3K activity (Wuttke et al 2010). Thus, the intensity of GFP at the cell
membrane would increase if GABA stimulated the PI3K pathway activity.
However, there was no increase in the GFP intensity when GABA was
added to the cells. The PI3K pathway seems therefore not to be involved in
the GABA-mediated maintenance of the proliferation of NPE cells.
36
´
Figure 10. Schematic diagram illustrating the proposed mechanism on how
embryonic depolarising GABAA receptors and L-type VGCCs regulate proliferation.
Activation of GABAA receptors generates a depolarised plasma membrane and
activation of L-type voltage-gated Ca2+ channels (VGCCs). The intracellular Ca2+
concentration increases and leads to up-regulation of immediate early genes
(Morgan & Curran 1988). c-Fos and c-Jun form the Activator Protein-1 (AP-1)
complex that represses the expression of cyclin-dependent protein kinase inhibitors
(p27KIP1) and cyclin-cyclin dependent kinases (CDKs) are free to promote cell cycle
progression. Growth factors work through the same pathway to stimulate the
expression of immediate early genes.
Paper IV
Retinal neurons are generated in a conserved temporal order during
development. Combinations of transcription factors such as Ath3, NeuroD1,
Pax6, Pax2 and Otx2 regulate the cell fates of RPCs (Akagi et al 2004a,
Hatakeyama et al 2001, Inoue et al 2002, Ohsawa & Kageyama 2008).
Wider knowledge about the different factors controlling specific cell fates is
needed in order to use stem- or progenitor cells for repairing an injured
retina. The transcription factor Forkhead box N4 (FoxN4) has been shown to
commit RPCs into certain cell fates. In a knockout mouse, lacking the
expression of FoxN4, all retinal HCs were lost and the number of ACs was
decreased whereas the number of PRs was increased (Li et al 2004).
Furthermore, the proliferation of RPCs was shown to decrease in the areas
37
where FoxN4 expression had been knocked out. In Paper IV, we aimed to
further investigate the function of FoxN4 in the retina by studying its native
expression and by over-expressing it in the developing chick retina.
Different antibodies that specify identities of different cell types were used
together with an antibody against FoxN4 to examine the role of the
transcription factor. FoxN4 was found to be expressed only by progenitors in
the developing retina. FoxN4-expressing cells in the optic vesicle were
expressing Pax2 (Fig. 1A-D in Paper IV), and throughout development
FoxN4 expressing RPCs weakly expressed Pax6 and strongly Sox2 (Fig. 2 in
Paper IV). Cells that expressed high levels of FoxN4 were expressing Lim1,
which is a marker of an early-generated subpopulation of HCs (Edqvist et al
2008) (Fig. 3A in Paper IV). Over-expression of FoxN4 resulted in
activation of gene markers of HCs, (such as Prox1 and Lim1), and the
number of HCs was increased compared to control eyes (Fig. 4F-G in Paper
IV). However, FoxN4 had not only effects on HCs but also on markers of
other retinal cell subtypes, such as PRs (Lim3 expression) and GCs (Brn3a
expression) (Fig. 11), were upregulated in areas where FoxN4 had been
over-expressed. Furthermore, we over-expressed FoxN4 in NPE cells. The
over-expression did not increase the expression of Lim1, Lim3 or Brn3a
(qRT-PCR and immunohistochemistry, data not shown). These results
suggest that the response of a progenitor cell, to FoxN4 expression, is
determined by the intrinsic properties of the cell.
In order to determine if FoxN4 is able to trigger differentiation in the
developing CNS in general, FoxN4 was over-expressed in the neural tube of
embryonic chickens. The over-expression of FoxN4 induced expression of
many cell-specific gene markers (Fig. 6 in Paper IV).
Our study suggests that the effects of FoxN4 on progenitor cells depend
on the specific competence of the cells. Some cells have the competence to
respond to the expression of FoxN4 and can, dependent on their specific
properties, differentiate towards certain cell types. We conclude that FoxN4
is involved in the generation of HCs but that FoxN4 promotes cell specificity
dependent on the specific characteristics of the progenitor cells.
Luo and colleagues (Luo et al 2012) recently suggested that the Notch
ligand Delta like 4 (Dll4) is involved in the FoxN4-induced generation of
HCs and ACs in the mouse retina. Loss of FoxN4 function suppresses HCand AC fates and promotes PR fate (Li et al 2004). FoxN4-null retinas were
shown in a micro array analysis to down-regulate the expression of
transcription factors involved in specification of HCs and ACs, for example
Ptf1a, NeuroD1, NeuroD4 and Prox1; and genes involved in the Notch
signalling pathway, such as and Dll4 and Hes5. Over-expression of FoxN4
increased the expression of Dll4 and genetic ablation of Dll4 lead to an
increased PR production. In our study, we analysed the relative mRNA
levels of Dll4 in chicken retinas electroporated with FoxN4 but there was no
difference compared to control. Thus, Dll4 seems not to be involved in the
38
FoxN4-induced differentiation of chicken RPCs. Furthermore, we analysed
if the CDK inhibitor p27KIP1 was regulated by FoxN4 but there was no
difference between the FoxN4 over-expressed retinas and the control retinas.
Thus, currently we do not know the mechanism behind FoxN4-triggered
differentiation but the competence within the RPC seems to be crucial.
Figure 11. Over-expression of FoxN4 at st20 followed by analysis after 24 h by (A)
Lim1, (B) Prox1, (C) Lim3 and (D) Brn3a immunoreactivity. Boxes indicate regions
with higher magnifications shown in single-channel epifluorescence. Double arrows:
FoxN4-GFP and the indicated antibody double labelling. pgcl, prospective ganglion
cell layer.
39
Conclusions
This thesis has focused on two important aspects of retinal repair:
proliferation and differentiation. Knowledge about how to expand the stemor progenitor cell pool is crucial for replacing injured or degenerated retinal
neurons. There is a growing body of evidence that many neurotransmitters
are involved in the proliferation of different cell types (Andäng et al 2008,
Berg et al 2011, Fiszman et al 1999, Kralj-Hans et al 2006, Luk et al 2003,
Ma et al 2000, Martins et al 2006, Ohtani et al 2003, Pearson et al 2002,
Schwirtlich et al 2010, Toriumi et al 2012, Wang & Kriegstein 2009, Vitalis
& Parnavelas 2003, Young & Cepko 2004). The major result in this thesis is
that GABA contributes to and maintains RPC proliferation.
Marie Öhman and colleagues previously showed that the transcript
encoding the α3 subunit of the GABAA receptor was subjected to A-to-I
RNA editing (Ohlson et al 2007). Our results showed that editing of the α3
transcript occurred in the chicken retina. The frequency of editing increased
after st35, corresponding to E8-9. Among 16 GABAA receptor subunits, all
but β1 and π subunits, had expression elevated above background levels
(Fig. 3A and 3B in Paper II) and the majority of the subunits had increased
mRNA expression levels after st35. This general increase in GABAA
receptor subunit expression occurred concomitantly with an increase of the
chloride co-transporter KCC2, which is generally thought to contribute to the
shift of the GABA system from excitatory (depolarising) to inhibitory
(hyperpolarising). Altogether, these results suggest that GABA causes cell
membrane depolarisation at least until st35. Furthermore, A-to-I RNA
editing has been shown to alter the GABA sensitivity and to generate
receptors with slower activation and faster deactivation rates (Nimmich et al
2009, Rula et al 2008). The non-edited α3 subunit expressed during CNS
development, when GABA is depolarising, may therefore mediate fastactivated and long-lasting excitatory responses (Rula et al 2008). The
depolarising currents mediated by GABA are important for a number of
developmental processes, including proliferation (Ben-Ari et al 2007). We
therefore investigated if GABA was involved in the proliferation of RPCs.
NPE cells were used to monitor the effects of GABA due to their
homogeneity and similarities to early RPCs (Fischer & Bongini 2010,
Fischer & Reh 2003). The results showed that the NPE cells express
functional extrasynaptic-like GABAA receptors that are involved in
regulating the proliferation of the cells. Inhibition of GABAA receptors
40
decreased the proliferation of dissociated NPE cells. We suggest that GABA
causes depolarisation of the cells and thereby activates L-type voltage gated
Ca2+ channels (VGCCs). This suggestion is based on: 1. the effects on
proliferation by the GABAA receptor antagonists could be counteracted by
addition of extracellular KCl (Fig. 2 in Paper III), a treatment that
depolarises the plasma membrane (Purves 2012), and 2. the expression of
KCC2 was relative low suggesting that GABA mediates excitatory currents
(Ben-Ari et al 2007). Furthermore, inhibition of the GABAA receptors or
VGCCs caused an increased expression of the CDK inhibitor p27KIP1. This in
turn suggests that GABAA, via VGCCs, interacts with components of the
cell cycle. In vivo, NPE cells are contact inhibited and mitotically quiescent,
which has been shown for retinal pigment epithelium and most other
epithelia (Kokkinopoulos et al 2011, Tamiya et al 2010). Proliferation of
NPE cells can be triggered by retinal damage or degeneration (Ducournau et
al 2011, Johnsen et al 2012) and by exogenous growth factors (Fischer &
Reh 2003). Our results show that also GABA is involved in the proliferation
of NPE cells. We further suggest that GABA is important for maintaining
the proliferation of RPCs in the retina since GABAA receptor inhibition
decreased the proliferation of progenitors in the intact st35 retina (E8-9; Fig.
3 in Paper III). At st35, the depolarising action by GABA reaches a peak
(Catsicas & Mobbs 2001), which further suggests that depolarisation of
progenitors by GABA is important for maintaining the proliferation.
Cell differentiation is a complex process involving both intrinsic- and
extrinsic factors. Transcription factors including members of the basic-loophelix (bHLH), homeodomain and forkhead families have been reported to be
intrinsic regulators of retinal differentiation. Some examples are Otx2 and
Crx that are required for differentiation of PRs (Chen et al 1997, Nishida et
al 2003); and Ptf1a, Prox1, Sall3 and FoxN4 for HCs (de Melo et al 2011,
Dyer et al 2003, Fujitani et al 2006, Li et al 2004). We aimed to further
clarify the role of FoxN4 in RPCs and to analyse if FoxN4 is sufficient to
commit cells to the HC fate in the chicken retina. During initial
retinogenesis, cells with high expression of FoxN4 co-labelled for Lim1
suggesting a commitment to the HC fate. Over-expression of FoxN4 in the
developing chick retina activated the HC-marker genes Prox1 and Lim1
suggesting an increased number of HCs. In addition, FoxN4 over-expression
initiated expressions of genes associated with PRs and GCs. However, overexpression in NPE cells did not activate expression of genes associated with
HCs, PRs or GCs. Altogether, these results indicate that the effects of FoxN4
on differentiation are dependent on the properties of the cell. The results
further suggest that the NPE cells are not a cellular source to use for
generating HCs, PRs or GCs by the means of FoxN4 over-expression. The
chicken NPE cells are able to generate neurons, such as GCs and ACs, in the
presence of growth factors (Fischer & Reh 2003). Our results indicate that
FoxN4, alone, is not sufficient to trigger this cell generation.
41
MCs can be stimulated to produce neurons in different classes of
vertebrates (Fischer & Bongini 2010). However, the MCs provide structural
support, synaptic support, osmotic homeostasis, and metabolic support to
retinal neurons (Fischer & Bongini 2010). Therefore, a damage-induced dedifferentiation and re-entry into the cell cycle of the MCs may be unsuitable
(Wohl et al 2012). This might be reflected by the fact that the majority of
cells produced by MC-derived progenitors do not differentiate into neurons,
but remains as progenitor-like cells or form new glia (Fischer et al 2002b,
Fischer & Reh 2001). Our results showed that MCs located in the central
retina express the transcription factor Pax2. The centrally located MCs are
less plastic, proliferative and neurogenic compared to MCs in peripheral
regions of the retina (Fischer 2005). The Pax2 expression was neither
increased nor decreased by induced injury. This suggests that Pax2 is not
involved in the de-differentiation or proliferation responses but rather in
maintaining the MCs as glial cells. Therefore, we suggest that expression of
Pax2 in MCs is important to maintain critical supportive functions to retinal
neurons. By maintaining these functions, Pax2 acts as a neuroprotective
factor in case of retinal injury.
The main aim of this thesis was to find and characterise different factors
involved in the proliferation and differentiation of RPCs and hence,
contribute to the knowledge how to use progenitor cells for retinal repair.
The results indicate that the different cell sources to use for retinal repair are
highly diverse in their potential to proliferate and produce neurons. We
conclude that GABA, Pax2 and FoxN4 may be factors to consider when
designing strategies for retinal repair. However, our results indicate that the
response to these factors is highly associated with the specific properties of
the progenitor cells.
42
Material and Methods
Animals
All experiments in Papers I-IV were carried out in strict accordance with the
recommendation in the Guide for the Care and Use of Laboratory Animals
of the Association for Research in Vision and Ophthalmology. The protocols
were approved by the Committee on the Ethics of Animal Experiments by
Uppsala djurförsöketiska nämnd.
Fertilised White Leghorn eggs were obtained from Ova Production AB
(Västerås, Sweden) and incubated at 38oC in a humidified incubator.
Embryos were staged (st) according to Hamburger and Hamilton
(Hamburger & Hamilton 1951).
Immunohistochemistry
Eyes were fixed in 4% paraformaldehyde in PBS for 15 minutes at 4oC,
incubated for 3 hours in 30% phosphate-buffered sucrose at 4oC, embedded
in OCT freezing medium (Tissue-Tek), frozen and sectioned in a cryostat.
Retinas were cryosectioned sagittally through the lens producing 10 µm
dorsal to ventral sections of the retina. Primary antibodies were incubated
over night at 4oC, and secondary antibody for 2 hours at room temperature.
Quantitative reverse transcription PCR
Total RNA was isolated from cells or tissue by using TRIzol reagent
(Invitrogen). Four RNA preparations from cells or tissue were collected.
Complementary DNA (cDNA) was prepared from 1 μg of RNA using
GeneAmp (Applied Biosystems).
The quantitative reverse transcription PCR (qRT-PCR) analyses were
performed using IQ™ SyBr Green Supermix (Biorad) with primers designed
by using Primer Express v2.0, default setting; Tm 60°C, 50% G/C, and
amplicon size minimum 100 base pairs. Each primer sequence was blasted
separately against GenBank and EMBL and only primers with a perfect
match within the target sequence and with the second best hit <75% identity,
were used. To confirm identity of amplified PCR products, dissociation
43
curve analyses and agarose gel electrophoresis were performed. The initial
mRNA levels were normalised to β-actin and TATA-box binding protein
(TBP) mRNA levels in order to correct variations in the cDNA syntheses.
Proliferation analyses: EdU assay, [3H]-thymidine
incorporation and MTT assay
Click iT EdU imaging kit (Invitrogen) was used to visualize cells passing
through S-phase (Paper I and III). In Paper I the thymidine analogue 5ethynyl-2’-deoxyuridine (EdU) was injected into the yolk of embryos and in
Paper III EdU was added to cell cultures. The EdU (Fig. 12A) was detected
according to the manufacturer’s protocol and EdU positive and negative
cells were manually counted by using a Zeiss Axioplan2 microscope
equipped with Axiovision software (Carl Zeiss Vision GmbH).
In Paper III cells were treated with reagents together with [3H]-thymidine
to measure DNA synthesis. [3H]-thymidine (Fig 12A) incorporation was
examined (4 biological replicates per treatment) by harvesting the cells to a
glass fibre filtermat (Perkin-Elmer). This was analysed in a Wallac 1205
Betaplate Liquid Scintillation counter (Wallac).
Some of the results in Paper III were verified by using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; SigmaAldrich) assay. The MTT assay (Fig 12B) was performed according to the
manufacturer’s protocol and analysed on a Multiskan MS plate reader
(Labsystems).
44
Figure 12. Analyses of cell proliferation and viability. (A) Both EdU and [3H]thymidine are incorporated in the replicated DNA during cell proliferation. Thus,
incorporation of either of these thymidine analogues is proportional to the level of
cell proliferation. EdU is detected by a copper catalyzed covalent reaction between
an alkyne in the EdU and an azide. The azide is associated with a fluorophore, which
enables detection of cells that gone through S-phase by for instance fluorescent
microscopy. The incorporated isotope [3H]-thymidine is detected by its β radiation,
which can be measured in a scintillation counter. (B) MTT assay is often used to
measure cell viability or cell number. The yellow coloured tetrazol MTT is taken up
by cells and accumulates in mitochondria where it is reduced by succinate
dehydrogenease into a purple coloured formazan product. The intensity of the
formazan product can be measured spectrophotometrically. Since the reduction of
MTT only occurs in metabolic active cells, the level of activity is a measure of the
viability of the cells.
Flow cytometry
Fluorescence-activated cell sorting (FACS) was used in Paper III to study
the distribution of cells in the different phases of the cell cycle as well as
apoptosis among cells. Dissociated cells were treated with different
chemicals over night and fixed in 80% methanol. The cells were either
directly stained with 50 μg/ml propidium iodide (PI, Sigma-Aldrich, 0.1%
Triton X100 and 20 μg/ml RNase A in phosphate-buffered saline (PBS) or
treated with DNA extraction buffer (0.2 M Na2HPO4 and 0.1 M citric acid)
and then stained with PI. The samples were run on a BD LSR II flow
cytometer (BD Biosciences) using FACSDiva version 6.0 software, and
45
analysed by the ModFit LT DNA analysis software (Verity Software house;
version 3.2.1; Model 1Dn0n-DSD). The ModFit DNA analysis was based on
12 000 events/treatment (4 biological replicates/treatment). Fragmented
DNA in apoptotic cells with less DNA than G1 cells was detected by FACS
analysis.
Electrophysiology
Whole-cell patch-clamp recordings were performed in Paper III to analyse
the presence of functional GABAA receptors on freshly dissected NPE cells.
The cells were washed with extracellular recording solution containing in
mM: 145 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2 and 10 TES pH 7.4. GABA and
SR-95531 were dissolved in the extracellular recording solution. The cells
were treated with either 1 µM or 100 µM of GABA. The pipette solution
contained in mM: 125 KCl, 5 CsCl, 1 MgCl2, 1.8 CaCl2, 5 EGTA and 10
TES pH 7.4. The pipette holding potential was -90 mV. Pipettes were made
of borosilicate glass and the pipette resistance used for the whole-cell
recordings ranged from 4 to 8 mΩ. The average current was measured as the
average of deviation of all data points from the middle of the baseline
current. The whole-cell currents were recorded with an Axopatch 200B
amplifier (Molecular devices), filtered at 2 kHz, digitised at 10 kHz using a
digiData 1322A analogue-to-digital converter interfaced with a computer
and analysed by pClamp 9.2 software.
In situ hybridization
Dissected eyes were fixed in 4% paraformaldehyde in phosphate-buffered
saline for 1 hour at 4oC. Fixed eyes were then incubated overnight in 30%
phosphate-buffered sucrose at 4oC, embedded in OCT freezing medium
(Tissue-Tek), frozen and sectioned in a cryostat. Ten µm thick sections
transversely cut through the optic nerve were collected on glass slides (Super
Frost Plus, Menzel-Gläser). In situ hybridization analysis (Paper II) was
performed as previously described (Boije et al 2008, Pringle 2006). In short,
a cRNA probe was made using the DIG RNA labelling kit (Roche). Probes
were hybridized to untreated sections over night at 68oC under conditions
containing 50% formamide and 5X SSC in a humidified chamber. The DIG
labelled probes were detected by using an alkaline-phosphatase conjugated
anti-DIG antibody (Roche) followed by incubation with BCIP/NBT
developing solution (Roche) for 2-5 hrs at 37oC. Images were captured by
using a Zeiss Axioplan2 microscope equipped with Axiovision software
(4.8, Carl Zeiss Vision GmbH).
46
Sequencing
In Paper II, the extent of editing of the transcripts for the GABAA receptor
subunit α3 was determined by sequencing of cDNA from different
embryonic ages and adult chicken. RNA was isolated by using TRIzol
(Invitrogen). Four RNA preparations were collected. Random primers were
used for first-strand cDNA synthesis (Superscript III RT, Invitrogen). PCR
(Platinum Taq, Invitrogen) was carried out with primers specific for α3
subunit (GABRA3) sequences. The PCR products were gel-purified and
sequenced and the relative peak heights in the chromatogram were compared
for A/G at nucleotide position 10884709 in GABRA3 on chick chromosome
4 (WashUC2), corresponding to exon 8 and amino acid 5 in the third
transmembrane domain of the α3 subunit. Pyrosequencing (Fig. 13) was
used to verify the RNA editing. Four parallel PCR reactions (1 PCR
reaction/cDNA batch) with an unbiotinylated forward primer and a
biotinylated reverse primer were carried out producing 121 base pairs PCR
fragments covering the edited nucleotide in chick α3 subunit sequence. The
biotinylated PCR products were bound to streptavidin sepharose high
performance beads (GE Healthcare) and denatured before annealing
(Qiagen) to a specific sequencing primer. Sequencing reactions were run on
a PSQ96 MA pyrosequencing machine using pyroMark Gold Q96 reagents
(Qiagen). The Pyrosequencing analysis is designed to differentiate between
specific single nucleotide polymorphisms at defined positions, and we tested
for the presence of A at nucleotide position 10884709, which corresponds to
the unedited variant of the GABRA3 transcript. To quantify the editing, PCR
products from cDNA made from a pool of all stage specific RNA samples
were cloned in pGEM-T easy vectors (Promega) and 20 clones originating
each stage were individually sequenced. Chick genomic DNA was
sequenced as a control.
47
Figure 13. Preparation and running of pyrosequencing used for examination of RNA
edited transcripts. cDNA is made from RNA prepared from tissue or cells of interest.
The cDNA is used as template in a PCR reaction including a biotinylated primer and
an unbiotinylated primer producing PCR fragments covering the edited nucleotide.
The biotinylated PCR products are bound by streptavidin beads and denaturated in
order to anneal to a specific sequencing primer designed to be reversed to the
biotinylated primer and not to be more than five nucleotides away from the edited
nucleotide. In pyrosequencing four enzymes, DNA polymerase, ATP sulfurylase,
luciferase and apyrase, convert synthesis of a complementary DNA strand into light
signals, which expose if a specific nucleotide is present or not in the biotinylated
PCR template sequence. The pyrosequencing machine tests if one of the four
nucleotides, A, C, G or T, is able to bind complementary to the biotinylated PCR
template. Incorporation will result in a chemical reaction producing a light signal,
which is shown on a pyrogram.
Electroporation
In Paper IV pCIG vectors carrying FoxN4-IRES-nls-GFP or nls-GFP under
a β-actin promoter were injected at a concentration of 4 µg/µl into either the
lumen of the brain vesicles at st13 (E2) or subretinally at st20 (E3).
Electrodes were placed at both sides of the head, and electroporation was
carried out using a BTX ECM830 electroporator delivering five 50 ms
pulses of 18V. Transfected embryos were allowed to develop for 24h, 48h or
until st35 (E9) before analysis.
48
Acknowledgements
I would like thank my supervisor Finn Hallböök, for giving me the
opportunity and liberty to explore science without too many harsh directives.
Sometimes it has been quite frustrating to get instructions such as “can you
have a look on this gene”, without any other comments. However, I am sure
that such “short-spoken” instructions have made me a better PhD student.
After all, science is about (questioning and) finding answers. I appreciate
that you have encouraged me to participate in other activities, for example as
Ph.D. student representative on the Board of the department of
Neuroscience. Thank you also for the summer and Christmas parties! I thank
my co-supervisor, Per Söderberg, for interesting discussions (especially
about the lab instruments that you designed and built yourself)!
Henrik Boije, what should I say...? You are one of kind and I think all of
us have missed your sense of humour since you left the retina group. I will
probably always remember our conferences including “I’m a frog”, “it’s cold
in the sea”, “use your elbows” etcetera. I miss our Friday afternoon sessions
of YouTube movies: ”Jag svänger ju inte”, "Vad skulle Eva Dahlgren ha
gjort i det här läget? Asså om hon vore straight." Thank you for all our
scientific discussions and your good advice as well!
Shahrzad and Maria, thanks for nice lunch conversations and teaching
activities. Do not worry; I think everything will be just fine. Shahrzad, please
do not repeat my experiments. I do not want to get an e-mail a couple of
years from now telling me that I misinterpreted my results. Miguel, we do
not say that much to each other, although we share room, but I have really
enjoyed the times we talked about research or the (bad) weather . Big
thanks to the other present members of the retina group: Rashid and Niclas,
and to the former members: Ulrika, Robert and Madelen.
Thanks to the students I have had the pleasure to supervise, Linnea, Ada,
Alexandra, Björn and Per.
I would also like to thank:
Ebendal group: Ted, Annika, Lotta and Nestor for helping me with
different issues, especially cell work-related ones. Danlab and
Aldskogius/Kozlova groups. Bryndis Birnir and Suresh Kumar Mendu
for nice collaboration and interesting discussions. Klas Kullander group,
especially Fatima and Christiane for introducing me to animals that are not
chickens. Christine (Anki) Lindström at the Department of Genetics and
Pathology for helping me with the pyrosequencing, and Lisbeth Fuxler at
49
the Department of Biomedicial Sciences and Veterinary Public Health for
helping me with cell harvesting and β counting. Nigel for being a good
friend and for reading and commenting my English without using the red
pen too often. My parents for being supportive, even though I need to
explain what I am doing every time we talk about my work. I am happy to
have you as my parents!
Special thanks to my beautiful wife Claudia, for always being supportive
and showing interest in my work even though you sometimes find it a bit
boring. You are always here for me and I am here for you.
If I had been PH (Edqvist) I would probably thank the lunch lady down
in the cafeteria as well, but I am not and will therefore end my list of people
I like to thank by this sentence .
50
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Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 821
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala
University, is usually a summary of a number of papers. A few
copies of the complete dissertation are kept at major Swedish
research libraries, while the summary alone is distributed
internationally through the series Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of
Medicine.
Distribution: publications.uu.se
urn:nbn:se:uu:diva-180011
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2012