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Generation of Retinal Neurons: Focus on the Proliferation and Differentiation of the Horizontal Cells and their Subtypes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

We have used the chicken retina as a model for investigating cell cycle regulation and cell fate commitment during central nervous system development. This thesis focuses on the characterization of and commitment to the horizontal cell fate in the retina. Horizontal cells are interneurons that provide intraretinal signal processing prior to information relay to the brain. We have identified molecular markers that selectively distinguish the three subtypes of horizontal cells, previously described in the chicken retina based on morphology. Subtype specific birth-dating revealed that horizontal cell subtypes are generated consecutively by biased progenitors that are sensitive to the inhibitory effects of follistatin. Follistatin stimulates proliferation in progenitors by repressing the differentiation signal of activin. Initially, injection of follistatin led to a decrease in committed horizontal cells but as the inhibitory effect dissipated it resulted in an increased number of horizontal cells. During development committed horizontal cell progenitors migrate to the vitreal side of the retina where they become arrested in G2-phase for approximately two days. When the arrest is overcome the horizontal cell progenitors undergo ectopic mitosis followed by migration to their designated layer. The G2-phase arrest is not triggered or maintained by any of the classic G2-arrest pathways such as DNA damage or stress. Nevertheless, we show that the cyclin B1-Cdk1 complex has a central role in maintaining this G2-phase arrest. Two transcription factors, FoxN4 and Ptf1a, are required for the generation of horizontal cells. We show that these factors are also sufficient to promote horizontal cell fate. Overexpression of FoxN4 and Ptf1a resulted in an overproduction of horizontal- and amacrine cells at the expense of ganglion- and photoreceptor cells. We identified Atoh7, a transcription factor required for the generation of ganglion cells, as a Ptf1a transcriptional target for downregulation. Our data support a common horizontal/amacrine lineage separated from the ganglion/photoreceptor lineage by the action of Ptf1a. In conclusion, these data describe several novel characteristics of horizontal cells enhancing our understanding of neural development and cell fate commitment.

Place, publisher, year, edition, pages
Uppsala: Acta Universitalis Upsaliensis , 2011. , 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 672
Keyword [en]
FoxN4, Ptf1a, PH3, G2-phase, Cell cycle arrest, Differentiation, Fate, Commitment, Neurogenesis, Follistatin
National Category
Neurosciences
Research subject
Neuroscience
Identifiers
URN: urn:nbn:se:uu:diva-150886ISBN: 978-91-554-8074-5OAI: oai:DiVA.org:uu-150886DiVA: diva2:409969
Public defence
2011-05-28, B42, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2011-05-06 Created: 2011-04-07 Last updated: 2011-07-01Bibliographically approved
List of papers
1. Axon-bearing and axon-less horizontal cell subtypes are generated consecutively during chick retinal development from progenitors that are sensitive to follistatin
Open this publication in new window or tab >>Axon-bearing and axon-less horizontal cell subtypes are generated consecutively during chick retinal development from progenitors that are sensitive to follistatin
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2008 (English)In: BMC Developmental Biology, ISSN 1471-213X, Vol. 8, 46- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Horizontal cells are retinal interneurons that modulate the output from photoreceptors. A rich literature on the morphological classification and functional properties of HCs in different animals exists, however, the understanding of the events underlying their development is still limited. In most vertebrates including chicken, two main horizontal cell (HC) subtypes are identified based on the presence or absence of an axon.

RESULTS:

In this work we have molecularly characterized three HC subtypes based on Lim1, Isl1, GABA and TrkA, a classification that is consistent with three chick HC subtypes previously defined by morphology. The axon-bearing and axon-less HC subpopulations molecularly defined by Lim1 and Isl1, are born consecutively on embryonic day (E) 3-4 and E4-5, respectively, and exhibit temporally distinguishable periods of migration. Their relative numbers are not adjusted by apoptosis. A sharp decrease of high endogenous levels of the activin-inhibitor follistatin at E3 coincides with the appearance of the Lim1 positive cells. Extending the follistatin exposure of the HC retinal progenitor cells by injection of follistatin at E3 increased the number of both Lim1- and Isl1 positive HCs when analysed at E9.

CONCLUSION:

The results imply that the axon-bearing and axon-less HC subgroups are defined early and are generated consecutively from a retinal progenitor cell population that is sensitive to the inhibitory action of follistatin. The results are consistent with a model wherein added follistatin causes HC-generating progenitors to proliferate beyond the normal period of HC generation, thus producing extra HCs of both types that migrate to the HC layer.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-102172 (URN)10.1186/1471-213X-8-46 (DOI)000255933400001 ()18439241 (PubMedID)
Available from: 2009-05-05 Created: 2009-05-05 Last updated: 2012-03-09Bibliographically approved
2. Horizontal cell progenitors arrest in G2-phase and undergo terminal mitosis on the vitreal side of the chick retina
Open this publication in new window or tab >>Horizontal cell progenitors arrest in G2-phase and undergo terminal mitosis on the vitreal side of the chick retina
2009 (English)In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 330, no 1, 105-113 p.Article in journal (Refereed) Published
Abstract [en]

We have addressed the question when horizontal cells in the chick retina are generated and undergo their terminal mitosis. Horizontal cell progenitors replicate their DNA early and migrate bi-directionally to the horizontal cell layer. It was hypothesized that the cells undergo mitosis directly after replication and migrate as post-mitotic transition cells before differentiating to horizontal cells. However, our results show that cells expressing markers for the axon-bearing and the axon-less subtypes of horizontal cells undergo terminal mitosis while residing on the vitreal side of the retina. By combining horizontal cell transcription factors Lim1, Isl1 and Prox1 labeling with phospho-histone H3, a marker for mitosis, we demonstrate that all or a clear majority of vitreal mitoses are undertaken by the horizontal cell committed progenitors. The pattern of cells that incorporated the thymidine analogue EdU implied that the progenitors replicated their genome while migrating towards the vitreal side. Upon arrival to the vitreal retina they become arrested for about two days prior to mitosis. Hence, cells expressing horizontal cell markers are arrested in G2-phase on the vitreal side of the retina. These results support the existence of committed progenitors that give rise to horizontal cells and that those cells become arrested in G2-phase before undergoing terminal mitosis on the vitreal side of the retina followed by migration to the horizontal cell layer. The results also indicate that the regulation of the transition from G2-phase to mitosis is important for the development of these committed progenitor cells.

Place, publisher, year, edition, pages
Elsevier, 2009
Keyword
Cell cycle, Differentiation, Development, EdU, Prox1, Lim1, Stem cell
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-120381 (URN)10.1016/j.ydbio.2009.03.013 (DOI)000266300800010 ()19324032 (PubMedID)
Available from: 2010-03-11 Created: 2010-03-11 Last updated: 2011-07-01Bibliographically approved
3. The terminal mitosis of chicken retinal horizontal cells is preceded by a G2-Phase arrest that relies on the cyclin B1-Cdk1 complex but is independent of DNA damage.
Open this publication in new window or tab >>The terminal mitosis of chicken retinal horizontal cells is preceded by a G2-Phase arrest that relies on the cyclin B1-Cdk1 complex but is independent of DNA damage.
(English)Article in journal (Other academic) Submitted
Identifiers
urn:nbn:se:uu:diva-150879 (URN)
Available from: 2011-04-11 Created: 2011-04-07 Last updated: 2011-07-01
4. Temporal and spatial expression of transcription factors FoxN4, Ptf1a, Prox1, Isl1 and Lim1 mRNA in the developing chick retina
Open this publication in new window or tab >>Temporal and spatial expression of transcription factors FoxN4, Ptf1a, Prox1, Isl1 and Lim1 mRNA in the developing chick retina
2008 (English)In: Gene Expression Patterns, ISSN 1567-133X, E-ISSN 1872-7298, Vol. 8, no 2, 117-123 p.Article in journal (Refereed) Published
Abstract [en]

Transcription factors are pivotal in regulating cell fate and development. We analyzed five transcription factors - FoxN4, Ptf1a, Prox1, Isl1 and Lim1 - with putative functions in the formation of early-generated retinal interneurons. A full-length chicken FoxN4 cDNA was characterized and in situ as well as RT-PCR showed that FoxN4 expression commenced already in the stage 12-14 optic vesicles. Ptf1a, Prox1, Isl1 and Lim1 expression appeared later by stage 20-24, concomitant with the first post-mitotic ganglion-, amacrine- and horizontal cells. The FoxN4 and Ptf1a expression was transient with peak levels by stage 32-35. Expression disappeared as the retinal progenitor cells differentiated. Prox1, Isl1 and Lim1 expression remained in several differentiated cells including the horizontal cells. The order of expression supports a scheme where Ptf1a and Prox1 is downstream of FoxN4 and that FoxN4 and Ptf1a have transient roles during fate specification while Prox1, Isl1 and Lim1 have roles that are important for the generation of the neuronal subtypes.

Keyword
In situ hybridization, Optic vesicle, Retina, Quantitative RT-PCR, RACE
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-87576 (URN)10.1016/j.modgep.2007.09.004 (DOI)000252714300009 ()18006384 (PubMedID)
Available from: 2009-01-05 Created: 2008-12-30 Last updated: 2012-03-09Bibliographically approved
5. Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina
Open this publication in new window or tab >>Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina
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2011 (English)In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 358, no 2, 296-308 p.Article in journal (Refereed) Published
Abstract [en]

During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix–loop–helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.

Keyword
Cell specification, Horizontal cell, Atoh7/Ath5, Retina; Chick
National Category
Medical and Health Sciences Basic Medicine
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-150878 (URN)10.1016/j.ydbio.2011.07.033 (DOI)000295603900003 ()21839069 (PubMedID)
Available from: 2011-04-09 Created: 2011-04-07 Last updated: 2015-08-13Bibliographically approved
6. FoxN4 is sufficient for commitment to the retinal horizontal cell fate and is able to instigate differentiation programs in neural progenitors
Open this publication in new window or tab >>FoxN4 is sufficient for commitment to the retinal horizontal cell fate and is able to instigate differentiation programs in neural progenitors
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:uu:diva-150880 (URN)
Available from: 2011-04-11 Created: 2011-04-07 Last updated: 2011-07-01

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