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Keeping up with retinal photoreceptors and horizontal cells: Labelling and mapping of cells in the normal and diseased embryonic chicken retina
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience. (Finn Hallböök)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The childhood eye cancer retinoblastoma originates from the retina and its development is initiated while the foetus is in the uterus. Retinoblastoma has a reported incidence of 1 in 15-18 000 live births, and approximately 90% of all patients are diagnosed before the age of 5. The occurrence of retinoblastoma is usually detected by the parents and the most frequent symptoms are leukocoria (white pupillary reflex), strabismus (squinting) or if the child complains of visual problems. Retinoblastoma is diagnosed by examination under anaesthesia and documentation by RetCam. It is treated with various cytostatic agents, or by laser. If the treatment is unsuccessful, or there is a risk that the tumour cells will spread and form metastases, the eye is removed.

Previous studies have indicated that the cell type from which the tumour arises, the cell-of-origin, may be the cone photoreceptors and/or their immediate interneuron, the horizontal cells. Determining the cell-of-origin for retinoblastoma is an important goal, however, understanding the molecular mechanisms that distinguish the photoreceptors and the horizontal cells from the other retinal cells may prove just as important for understanding this disease.

The aim of my project has been to develop, optimise and validate methods to label, map and target expression to photoreceptors and horizontal cells in the chicken embryonic retina. We have successfully established several methods that test the expression pattern of conserved, regulatory DNA sequences, and have performed short- and long-term expression of various genes that have been reported to be involved in cell cycle regulation and cell fate determination. One of my most important findings was that a region from the RXRγ gene allowed us to specifically target the photoreceptors and horizontal cells. Our previous knowledge, together with the newly established tools, puts us an important step closer towards understanding the development and behaviour of the retinal photoreceptors and horizontal cells, however, further studies are of course needed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 62
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1302
Keyword [en]
Chicken, electroporation, horizontal cells, photoreceptors, retina, piggyBac
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:uu:diva-315655ISBN: 978-91-554-9825-2 (print)OAI: oai:DiVA.org:uu-315655DiVA, id: diva2:1075060
Public defence
2017-04-07, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-03-10 Created: 2017-02-17 Last updated: 2018-01-13
List of papers
1. Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments
Open this publication in new window or tab >>Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments
2015 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 103, article id e53202Article in journal (Refereed) Published
Abstract [en]

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high. This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

Keyword
Developmental Biology, Issue 103, Culture, explant, eye, inhibitors, in ovo, in vitro, plasmid
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-268713 (URN)10.3791/53202 (DOI)000364222300048 ()
Funder
Swedish Childhood Cancer Foundation, PR2013-0104Swedish Research Council, 12187-18-3
Available from: 2015-12-09 Created: 2015-12-09 Last updated: 2018-01-10
2. The p53 co-activator Zac1 neither induces cell cycle arrest nor apoptosis in chicken Lim1 horizontal progenitor cells
Open this publication in new window or tab >>The p53 co-activator Zac1 neither induces cell cycle arrest nor apoptosis in chicken Lim1 horizontal progenitor cells
2015 (English)In: Cell Death Discovery, ISSN 2058-7716, Vol. 1, article id 15023Article in journal (Refereed) Published
Abstract [en]

Chicken horizontal progenitor cells are able to enter their final mitosis even in the presence of DNA damage despite having a functional p53-p21 system. This suggests that they are resistant to DNA damage and that the regulation of the final cell cycle of horizontal progenitor cells is independent of the p53-p21 system. The activity of p53 is regulated by positive and negative modulators, including the zinc finger containing transcription factor Zac1 (zinc finger protein that regulates apoptosis and cell cycle arrest). Zac1 interacts with and enhances the activity of p53, thereby inducing cell cycle arrest and apoptosis. In this work, we use a gain-of-function assay in which mouse Zac1 (mZac1) is overexpressed in chicken retinal progenitor cells to study the effect on the final cell cycle of horizontal progenitor cells. The results showed that overexpression of mZac1 induced expression of p21 in a p53-dependent way and arrested the cell cycle as well as triggered apoptosis in chicken non-horizontal retinal progenitor cells. The negative regulation of the cell cycle by mZac1 is consistent with its proposed role as a tumour-suppressor gene. However, the horizontal cells were not affected by mZac1 overexpression. They progressed into S- and late G2/M-phase despite overexpression of mZac1. The inability of mZac1 to arrest the cell cycle in horizontal progenitor cells support the notion that the horizontal cells are less sensitive to events that triggers the p53 system during their terminal and neurogenic cell cycle, compared with other retinal cells. These properties are associated with a cell that has a propensity to become neoplastic and thus with a cell that may develop retinoblastoma.

Place, publisher, year, edition, pages
Nature Publishing Group, 2015
National Category
Neurology Developmental Biology
Research subject
Neurology
Identifiers
urn:nbn:se:uu:diva-279034 (URN)10.1038/cddiscovery.2015.23 (DOI)
Available from: 2016-02-28 Created: 2016-02-28 Last updated: 2017-02-17Bibliographically approved
3. The zinc finger gene Nolz1 is controlled by retinoic acid and regulates the formation of chicken retinal progenitors and Lim3 expressing bipolar cells
Open this publication in new window or tab >>The zinc finger gene Nolz1 is controlled by retinoic acid and regulates the formation of chicken retinal progenitors and Lim3 expressing bipolar cells
(English)Article in journal (Refereed) Submitted
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-315608 (URN)
Available from: 2017-02-16 Created: 2017-02-16 Last updated: 2018-01-13
4. A regulatory sequence from the retinoid X receptor gamma gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina
Open this publication in new window or tab >>A regulatory sequence from the retinoid X receptor gamma gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina
2016 (English)In: Molecular Vision, ISSN 1090-0535, E-ISSN 1090-0535, Vol. 22, p. 1405-1420Article in journal (Refereed) Published
Abstract [en]

PURPOSE: Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. METHODS: A 208 bp gene regulatory sequence from the chicken retinoid X receptor gammagene (RXRgamma208) was used to drive Cre expression. RXRgamma is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. RESULTS: Cells that drove Cre expression from the regulatory RXRgamma208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRgamma208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRgamma gene expression in the early retina. CONCLUSIONS: In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRgamma-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

National Category
Developmental Biology
Identifiers
urn:nbn:se:uu:diva-313804 (URN)000393909400001 ()28003731 (PubMedID)
Funder
Swedish Research Council, MH521.2013.3346Swedish Childhood Cancer Foundation
Available from: 2017-01-24 Created: 2017-01-24 Last updated: 2017-11-29Bibliographically approved
5. Overexpression of MYCN in the developing chicken retina increases the generation of photoreceptor cells
Open this publication in new window or tab >>Overexpression of MYCN in the developing chicken retina increases the generation of photoreceptor cells
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-315611 (URN)
Available from: 2017-02-16 Created: 2017-02-16 Last updated: 2018-01-13

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