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Characterization of Retinal Progenitor Cells: Focus on Proliferation and the GABAA Receptor System
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 60 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 821
Keyword [en]
Regeneration, Proliferation, Neurotransmitters, Müller cells, Differentiation, Retinal repair, Neurogenesis
National Category
Neurosciences Other Basic Medicine
Research subject
Medical Science
URN: urn:nbn:se:uu:diva-180011ISBN: 978-91-554-8489-7OAI: diva2:559126
Public defence
2012-12-13, B41, BMC, Husargatan 3, Uppsala, 10:00 (English)

Doctor of Philosophy (Faculty of Medicine)

Available from: 2012-11-22 Created: 2012-08-28 Last updated: 2013-02-11Bibliographically approved
List of papers
1. Pax2 Is Expressed in a Subpopulation of Muller Cells in the Central Chick Retina
Open this publication in new window or tab >>Pax2 Is Expressed in a Subpopulation of Muller Cells in the Central Chick Retina
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2010 (English)In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 239, no 6, 1858-1866 p.Article in journal (Refereed) Published
Abstract [en]

Muller cells in the chick retina are generally thought to be a homogeneous population. We show that the transcription factor Pax2 is expressed by Muller cells in the central chick retina and its expression was first observed at stage 32 (embryonic day [E] 7.5). Birth-dating indicated that the majority of Pax2-positive Muller cells are generated between stage 29 and 33 (E5.5-E8). At stage 42 (E16), several Muller cell markers, such as Sox2 and 2M6, had reached the peripheral retina, while the Pax2 labeling extended approximately half-way. A similar pattern was maintained in the 6-month-old chicken. Neither the Pax2-positive nor the Pax2-negative Muller cells could be specifically associated to proliferative responses in the retina induced by growth factors or N-methyl-D-aspartate. Pax2 was not detected in Muller cells in mouse, rat, guinea-pig, rabbit, or pig retinas; but the zebrafish retina displayed a similar pattern of central Pax2-expressing Muller cells.

avascular, development, EdU, glia, Muller glia, regeneration, Sox2, zebrafish
National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-136282 (URN)10.1002/dvdy.22309 (DOI)000278761700027 ()
Available from: 2010-12-11 Created: 2010-12-11 Last updated: 2013-12-18Bibliographically approved
2. Increased A-to-I RNA editing of the transcript for GABAA receptor subunit α3 during chick retinal development
Open this publication in new window or tab >>Increased A-to-I RNA editing of the transcript for GABAA receptor subunit α3 during chick retinal development
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2010 (English)In: Visual Neuroscience, ISSN 0952-5238, E-ISSN 1469-8714, Vol. 27, no 5-6, 149-157 p.Article in journal (Refereed) Published
Abstract [en]

Adenosine-to-inosine (A-to-I) RNA editing is a cotranscriptional or posttranscriptional gene regulatory mechanism that increases the diversity of the proteome in the nervous system. Recently, the transcript for GABA type A receptor subunit α3 was found to be subjected to RNA editing. The aim of this study was to determine if editing of the chicken α3 subunit transcript occurs in the retina and if the editing is temporally regulated during development. We also raised the question if editing of the α3 transcript was temporally associated with the suggested developmental shift from excitation to inhibition in the GABA system. The editing frequency was studied by using Sanger and Pyrosequencing, and to monitor the temporal aspects, we studied the messenger RNA expression of the GABAA receptor subunits and chloride pumps, known to be involved in the switch. The results showed that the chick α3 subunit was subjected to RNA editing, and its expression was restricted to cells in the inner nuclear and ganglion cell layer in the retina. The extent of editing increased during development (after embryonic days 8–9) concomitantly with an increase of expression of the chloride pump KCC2. Expression of several GABAA receptor subunits known to mediate synaptic GABA actions was upregulated at this time. We conclude that editing of the chick GABAA subunit α3 transcript in chick retina gives rise to an amino acid change that may be of importance in the switch from excitatory to inhibitory receptors.

Chloride ion channel, GABA(A) subunits, GABA receptor, KCC2, mRNA expression, Posttranscriptional modification
National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-143655 (URN)10.1017/S0952523810000180 (DOI)000285477900002 ()20843408 (PubMedID)
Available from: 2011-01-24 Created: 2011-01-24 Last updated: 2013-12-18Bibliographically approved
3. GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium:      
Open this publication in new window or tab >>GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium:      
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2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 5, e36874- p.Article in journal (Refereed) Published
Abstract [en]

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca2+ channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca2+ in regulating the cell cycle. 

National Category
urn:nbn:se:uu:diva-172512 (URN)10.1371/journal.pone.0036874 (DOI)000305336100070 ()
Available from: 2012-04-11 Created: 2012-04-10 Last updated: 2013-02-11Bibliographically approved
4. Forkhead box N4 (FoxN4) triggers context-dependent differentiation in the developing chick retina and neural tube
Open this publication in new window or tab >>Forkhead box N4 (FoxN4) triggers context-dependent differentiation in the developing chick retina and neural tube
(English)In: Differentiation, ISSN 0301-4681, E-ISSN 1432-0436Article in journal (Refereed) Submitted
Chicken, retinal development, Lim1, Prox1, retinal progenitor cells, Sox2
National Category
Research subject
Developmental Neurosciences
urn:nbn:se:uu:diva-180007 (URN)
Available from: 2012-08-28 Created: 2012-08-28 Last updated: 2014-05-19Bibliographically approved

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