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- Blixt, Maria K. E., et al.
(författare)
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Zinc finger gene nolz1 regulates the formation of retinal progenitor cells and suppresses the Lim3/Lhx3 phenotype of retinal bipolar cells in chicken retina
- 2018
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Ingår i: Developmental Dynamics. - : WILEY. - 1058-8388 .- 1097-0177. ; 247:4, s. 630-641
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Tidskriftsartikel (refereegranskat)abstract
- Background: The zinc-finger transcription factor Nolz1 regulates spinal cord neuron development by interacting with the transcription factors Isl1, Lim1, and Lim3, which are also important for photoreceptors, horizontal and bipolar cells during retinal development. We, therefore, studied Nolz1 during retinal development.Results: Nolz1 expression was seen in two waves during development: one early (peak at embryonic day 3-4.5) in retinal progenitors and one late (embryonic day 8) in newly differentiated cells in the inner nuclear layer. Overexpression and knockdown showed that Nolz1 decreases proliferation and stimulates cell cycle withdrawal in retinal progenitors with effects on the generation of retinal ganglion cells, photoreceptors, and horizontal cells without triggering apoptosis. Overexpression of Nolz1 gave more p27 positive cells. Sustained overexpression of Nolz1 in the retina gave fewer Lim3/Lhx3 bipolar cells.Conclusions: We conclude that Nolz1 has multiple functions during development and suggest a mechanism in which Nolz1 initially regulates the proliferation state of the retinal progenitor cells and then acts as a repressor that suppresses the Lim3/Lhx3 bipolar cell phenotype at the time of bipolar cell differentiation.
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| 2. |
- Blixt, Maria, et al.
(författare)
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MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma
- 2022
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Ingår i: Oncogenesis. - : Springer Science and Business Media LLC. - 2157-9024. ; 11:1, s. 34-
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Tidskriftsartikel (refereegranskat)abstract
- Retinoblastoma is a rare, intraocular paediatric cancer that originates in the neural retina and is most frequently caused by bi-allelic loss of RB1 gene function. Other oncogenic mutations, such as amplification and increased expression of the MYCN gene, have been found even with proficient RB1 function. In this study, we investigated whether MYCN over-expression can drive carcinogenesis independently of RB1 loss-of-function mutations. The aim was to elucidate the events that result in carcinogenesis and identify the cancer cell-of-origin. We used the chicken retina, a well-established model for studying retinal neurogenesis, and established human embryonic stem cell-derived retinal organoids as model systems. We over-expressed MYCN by electroporation of piggyBac genome-integrating expression vectors. We found that over-expression of MYCN induced tumorigenic growth with high frequency in RB1-proficient chicken retinas and human organoids. In both systems, the tumorigenic cells expressed markers for undifferentiated cone photoreceptor/horizontal cell progenitors. The over-expression resulted in metastatic retinoblastoma within 7–9 weeks in chicken. Cells expressing MYCN could be grown in vitro and, when orthotopically injected, formed tumours that infiltrated the sclera and optic nerve and expressed markers for cone progenitors. Investigation of the tumour cell phenotype determined that the potential for neoplastic growth was embryonic stage-dependent and featured a cell-specific resistance to apoptosis in the cone/horizontal cell lineage, but not in ganglion or amacrine cells. We conclude that MYCN over-expression is sufficient to drive tumorigenesis and that a cell-specific resistance to apoptosis in the cone/horizontal cell lineage mediates the cancer phenotype.
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| 4. |
- Harun-Or-Rashid, Mohammad, et al.
(författare)
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Endothelin B Receptors on Primary Chicken Müller Cells and the Human MIO-M1 Müller Cell Line Activate ERK Signaling via Transactivation of Epidermal Growth Factor Receptors
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:12
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Tidskriftsartikel (refereegranskat)abstract
- Injury to the eye or retina triggers Muller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signaling are key regulators of these processes in Muller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Muller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Muller cells as well as the human Muller cell line MIO-M1. The Muller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Muller cells and the human Muller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173) EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-inhibitor (AG1478), EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001), consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Muller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in which endothelins among several other functions, serve as an injury-signal that regulate the gliotic response of Muller cells.
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| 5. |
- Harun-Or-Rashid, Mohammad, 1980-, et al.
(författare)
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The Endothelin B Receptor Transactivates Epidermal Growth Factor Receptors in primary chicken Müller cells and in MIO-M1 Human Müller Cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Injury to the nervous system elicits signals that trigger a variety of cellular responses. Injury to the retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate, proliferate, attain retinal progenitor properties and in some species generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signalling are key regulators of these processes in Müller cells. The complexity of the extracellular signals that modulate and control the process are not fully understood. In this work we studied whether endothelin receptor signalling can activate EGFR and ERK signalling in Müller cells. Endothelin expression is robustly up-regulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell-types. We treated chicken Müller cells in vivo, cultured primary chicken Müller cells and the human Müller cell line MIO-M1 with receptor agonists and enzyme blockers, and analyzed endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that both chicken and human Müller cells express endothelin receptor B. Stimulation by using the endothelin receptor B agonist IRL1620 caused Src-kinase mediated ligand-dependent and ligand-independent EGFR transactivation. The effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR inhibitor (AG1478) and by inhibitors to extracellular matrix metalloproteinases (GM6001). Our data outline a mechanism how injury-induced endothelins may modulate the Müller cell responses by transactivation of EGFRs. The data give support to a view in which endothelins, among several other functions, serve as an injury-signal that regulate the gliotic response of Müller cells.
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| 6. |
- Konjusha, Dardan, 1992-
(författare)
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Modulation of retinal progenitors : A bird’s-eye view of retinal regeneration and disease
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cell populations of the retina and their intricate organization provide us with one of the most important senses – vision. All retinal cell populations are derived from a common progenitor pool as a result of tight regulation of proliferation, differentiation, dedifferentiation, and programmed cell death. Dysregulation of these processes, or injury to the retina, can result in loss of vision or in certain cases even cancer – i.e. retinoblastoma. Understanding the mechanistic basis of these processes allows for modeling cancer and retinal regeneration. To this purpose, the embryonic chicken retina, and cultures thereof, were subjected to pharmacological intervention and modulation of gene expression. To validate findings in a human model, some studies were extended with the use of human cell cultures or retinal organoids derived from human embryonic stem cells. The focus was on the late events of retinal neurogenesis. In Paper I, we investigated endothelins as potential modulators of injury-induced retinal regeneration, which is performed by Müller cells in certain species. Injured Müller cells will dedifferentiate and return to the progenitor pool. We found that stimulation of the endothelin receptor induces dedifferentiation by transactivation of the epidermal growth factor receptor and subsequent activation of the MAPK-signaling pathway, in both chicken Müller cells and an immortalized cell line with Müller cell properties. Our findings show that endothelins have potential as possible regulators of the injury response and subsequent regeneration of lost neurons performed by Müller cells.In Paper II, the Nolz1 transcription factor and its regulation of retinal neurogenesis was explored. We show that Nolz1 acts as a negative regulator of the cell cycle in retinal progenitors, and hinders bipolar cell specification by Lim3 gene repression.In Paper III, we investigated the final neurogenic mitosis of the cone photoreceptor/horizontal cell progenitor (cPR/HC) lineage. MYCN-overexpression in a functional RB1 setting produced neoplastic growth in a cell-type and developmental-stage specific manner. The cPR/HC-lineage alone escaped apoptosis and continued proliferation both in human retinal organoids and embryonic chicken retina. Our findings have implications for the etiology of retinoblastoma and show that MYCN alone can induce cancerogenesis. The tumors arise as a result if intrinsic properties of the cPR/HC-lineage, which have not been observed in other retinal populations. Taken together, this thesis gives novel knowledge regarding the late events of retinal neurogenesis, cell-type specification, and the inherent properties of certain retinal progenitor lineages in the healthy and diseased retina.
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| 7. |
- Zhang, Hanzhao, et al.
(författare)
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Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour.
- 2023
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Ingår i: Cellular Oncology. - 2211-3428 .- 2211-3436.
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Retinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken.METHODS: MYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells.RESULTS: The expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis.CONCLUSION: In conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour.
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