| 1. |
- Blixt, Maria K. E., et al.
(författare)
-
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
-
Ingår i: Developmental Dynamics. - : WILEY. - 1058-8388 .- 1097-0177. ; 247:4, s. 630-641
-
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.
|
|
| 2. |
|
|
| 3. |
- Blixt, Maria, et al.
(författare)
-
MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma
- 2022
-
Ingår i: Oncogenesis. - : Springer Science and Business Media LLC. - 2157-9024. ; 11:1, s. 34-
-
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.
|
|
| 4. |
- Brusini, Irene, et al.
(författare)
-
Changes in brain architecture are consistent with altered fear processing in domestic rabbits
- 2018
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:28, s. 7380-7385
-
Tidskriftsartikel (refereegranskat)abstract
- The most characteristic feature of domestic animals is their change in behavior associated with selection for tameness. Here we show, using high-resolution brain magnetic resonance imaging in wild and domestic rabbits, that domestication reduced amygdala volume and enlarged medial prefrontal cortex volume, supporting that areas driving fear have lost volume while areas modulating negative affect have gained volume during domestication. In contrast to the localized gray matter alterations, white matter anisotropy was reduced in the corona radiata, corpus callosum, and the subcortical white matter. This suggests a compromised white matter structural integrity in projection and association fibers affecting both afferent and efferent neural flow, consistent with reduced neural processing. We propose that compared with their wild ancestors, domestic rabbits are less fearful and have an attenuated flight response because of these changes in brain architecture.
|
|
| 5. |
- Edwards, Steven J., et al.
(författare)
-
High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy
- 2020
-
Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional cell cultures are able to better mimic the physiology and cellular environments found in tissuesin vivocompared to cells grown in two dimensions. In order to study the structure and function of cells in 3-D cultures, light microscopy is frequently used. The preparation of 3-D cell cultures for light microscopy is often destructive, including physical sectioning of the samples, which can result in the loss of 3-D information. In order to probe the structure of 3-D cell cultures at high resolution, we have explored the use of expansion microscopy and compared it to a simple immersion clearing protocol. We provide a practical method for the study of spheroids, organoids and tumor-infiltrating immune cells at high resolution without the loss of spatial organization. Expanded samples are highly transparent, enabling high-resolution imaging over extended volumes by significantly reducing light scatter and absorption. In addition, the hydrogel-like nature of expanded samples enables homogenous antibody labeling of dense epitopes throughout the sample volume. The improved labeling and image quality achieved in expanded samples revealed details in the center of the organoid which were previously only observable following serial sectioning. In comparison to chemically cleared spheroids, the improved signal-to-background ratio of expanded samples greatly improved subsequent methods for image segmentation and analysis.
|
|
| 6. |
- Fard, Shahrzad Shirazi, et al.
(författare)
-
The p53 co-activator Zac1 neither induces cell cycle arrest nor apoptosis in chicken Lim1 horizontal progenitor cells
- 2015
-
Ingår i: Cell Death Discovery. - : Nature Publishing Group. - 2058-7716. ; 1
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 7. |
- Ghaderi Berntsson, Shala, 1964-, et al.
(författare)
-
Aniridia with PAX6 mutations and narcolepsy
- 2020
-
Ingår i: Journal of Sleep Research. - : Wiley. - 0962-1105 .- 1365-2869. ; 29:6
-
Tidskriftsartikel (refereegranskat)abstract
- PAX6 gene mutations cause a variety of eye and central nervous system (CNS) abnormalities. Aniridia is often accompanied by CNS abnormalities such as pineal gland atrophy or hypoplasia, leading to disturbed circadian rhythm and sleep disorders. Less is known on the coincidence of narcolepsy in this patient group. We aimed to find out whether the circadian rhythm or sleep-wake structure was affected in patients with aniridia. Four members of a family segregating with congenital aniridia in two generations were included in the study. The patients were subjected to genetic testing for a PAX6 mutation, multiple sleep latency test, whole-brain magnetic resonance imaging (MRI), hypocretin-1 in cerebrospinal fluid, and Human Leukocyte Antigen DQ beta1*06:02. All four members were heterozygous for the pathogenic c.959-1G>A mutation in the PAX6 gene. Sleep disturbance was observed in all family members. The index patient was diagnosed with narcolepsy. MRI showed a hypoplastic pineal gland in all members. We describe the first case of a patient with PAX6 haploinsufficiency, aniridia and pineal gland hypoplasia diagnosed with narcolepsy type-1, suggesting a complex sleep disorder pathogenesis.
|
|
| 8. |
- Harun-Or-Rashid, Mohammad, 1980-, et al.
(författare)
-
Alpha 2-Adrenergic Receptor Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling via Transactivation of EGF Receptors in the Human MIO-M1 Müller Cell Line
- 2019
-
Ingår i: Current Eye Research. - : Informa UK Limited. - 0271-3683 .- 1460-2202. ; 44:1, s. 34-45
-
Tidskriftsartikel (refereegranskat)abstract
- Purpose: Alpha 2-adrenergic receptor (α2-ADR) agonists are used clinically for a range of indications including reducing elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Animal experiments show that α2-ADR agonists attenuate the injury-induced Müller cell dedifferentiation by a mechanism that involves activation and regulation of extracellular signal-regulated kinase (ERK) 1/2 leading to transactivation of epidermal growth factor receptors (EGFRs). The purpose of this study was to study and corroborate the activation of this system in human cells.Material and Methods: The human Müller cell line MIO-M1 was treated with the α2A-ADR agonist brimonidine in combination with inhibitors for Src-kinase, EGFR-kinase, matrix metalloproteinase (MMP) as well as small interfering RNAs (siRNAs) for the EGFR. The cells were analyzed using immunocytochemistry, quantitative PCR and western blot techniques.Results: Our results show that human MIO-M1 cells express α2A-ADRs and that stimulation of these receptors caused a robust increase of ERK1/2 and protein kinase B (PKB/AKT) (Thr-308) phosphorylation in MIO-M1 cells. P-ERK1/2 and P-AKT (Thr-308) signaling was mediated by Src-kinase and associated with phosphorylation of tyrosine residue of epidermal growth factor receptor (P-EGFR Y1173). In addition, the agonist caused activation of MMPs. These effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-kinase inhibitor (AG1478), EGFR-siRNA and a MMP inhibitor (GM6001).Conclusion: The results confirm that this human Müller cell line responds to ADR stimulation with phosphorylation of ERK and AKT, which suggests that it is possible to pharmacologically target ADR to modulate the early events in human Müller cell dedifferentiation in a similar fashion as been shown for chicken Müller cells.Abbreviations: CRALBP: cellular retinaldehyde binding protein; EGFR: epidermal growth factor receptor; ERK1/2: extracellular signal-regulated kinase 1/2; GS: glutamine synthetase; GPCR: G protein-coupled receptor; IR: immunoreactivity; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; P-ERK1/2: phospho-ERK1/2; qRT-PCR: quantitative reverse transcriptase PCR
|
|
| 9. |
- Hellsand, Minas, 1989-
(författare)
-
Models of Retinal Development and Disease
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- For a model of a human disease to be valid and useful, it is important that key genotypic and phenotypic traits are shared between model system and human. The work in this thesis has been focused on generating new and characterizing spontaneous models of three genetic disorders affecting the retina: retinoblastoma, a childhood cancer with its origin in the fetal retina, Stargardt disease, a juvenile form of macular degeneration, and Bardet-Biedl syndrome, a pleiotropic ciliopathy featuring retinal degeneration.In Paper I, we generated two novel models of MYCN-driven retinoblastoma, in developing chicken retinas and in human retinal organoids, in order to identify its cell of origin and to determine whether MYCN is sufficient in driving tumorigenesis. We found that MYCN was indeed sufficient and that the expression was uniquely tolerated by the cone and horizontal cell progenitor which survived and proliferated anachronistically. The results from our models are in alignment with observations that MYCN-driven retinoblastoma results in a more anaplastic and aggressive form of the cancer and we propose that the fate-restricted progenitor for cones and horizontal cells is its cell of origin.In Paper II, we analyzed the effects of a novel, spontaneous model of ABCA4-mediated Stargardt disease in Labrador retrievers and found that dogs homozygous for the mutation exhibited visual impairment and photoreceptor degeneration. Cone photoreceptors were scarce and the retinal pigment epithelium autofluorescent, indicative of lipofuscin accumulation, a hallmark of Stargardt disease. The phenotype and its etiology in this model are akin to human Stargardt disease.In Paper III, we investigated whether a spontaneous mutation in the TTC8 gene resulted in syndromic Bardet-Biedl syndrome in golden retrievers. We found that, in addition to progressive photoreceptor degeneration and visual impairment, the TTC8 mutation resulted in pleiotropic clinical signs of Bardet-Biedl syndrome with great inter-individual variation, congruent with Bardet-Biedl syndrome in humans.We find that the models described in this thesis are representative of the corresponding genetic disorders in human and that they could be of value for hypothesis generation (in chicken), experimental gene therapy (in dog), and as a human system to test pharmacological interventions (retinal organoids).
|
|
| 10. |
- Konjusha, Dardan, 1992-
(författare)
-
Modulation of retinal progenitors : A bird’s-eye view of retinal regeneration and disease
- 2022
-
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.
|
|
