Finn Hallböök

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.

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.

In golden retriever dogs, a 1 bp deletion in the canine TTC8 gene has been shown to cause progressive retinal atrophy (PRA), the canine equivalent of retinitis pigmentosa. In humans, TTC8 is also implicated in Bardet–Biedl syndrome (BBS). To investigate if the affected dogs only exhibit a non-syndromic PRA or develop a syndromic ciliopathy similar to human BBS, we recruited 10 affected dogs to the study. The progression of PRA for two of the dogs was followed for 2 years, and a rigorous clinical characterization allowed a careful comparison with primary and secondary characteristics of human BBS. In addition to PRA, the dogs showed a spectrum of clinical and morphological signs similar to primary and secondary characteristics of human BBS patients, such as obesity, renal anomalies, sperm defects, and anosmia. We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-length TTC8 transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet–Biedl syndrome with heterogeneous clinical signs.

Domestication has resulted in immense phenotypic changes in animals despite their relatively short evolutionary history. The European rabbit is one of the most recently domesticated animals, but exhibits distinct morphological, physiological, and behavioral differences from their wild conspecifics. A previous study revealed that sequence variants with striking allele frequency differences between wild and domestic rabbits were enriched in conserved noncoding regions, in the vicinity of genes involved in nervous system development. This suggests that a large proportion of the genetic changes targeted by selection during domestication might affect gene regulation. Here, we generated RNA-sequencing data for four brain regions (amygdala, hypothalamus, hippocampus, and parietal/temporal cortex) sampled at birth and revealed hundreds of differentially expressed genes (DEGs) between wild and domestic rabbits. DEGs in amygdala were significantly enriched for genes associated with dopaminergic function and all 12 DEGs in this category showed higher expression in domestic rabbits. DEGs in hippocampus were enriched for genes associated with ciliary function, all 21 genes in this category showed lower expression in domestic rabbits. These results indicate an important role of dopamine signaling and ciliary function in the evolution of tameness during rabbit domestication. Our study shows that gene expression in specific pathways has been profoundly altered during domestication, but that the majority of genes showing differential expression in this study have not been the direct targets of selection.

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD. Stargardt disease (STGD) is the most common inherited retinal disease causing visual impairment and blindness in children and young adults, affecting 1 in 8–10 thousand people. For other inherited retinal diseases, the dog has become an established comparative animal model, both for identifying the underlying genetic causes and for developing new treatment methods. To date, there is no standard treatment for STGD and the only available animal model to study the disease is the mouse. As a nocturnal animal, the morphology of the mouse eye differs from humans and therefore the mouse model is not ideal for developing methods for treatment. We have studied a novel form of retinal degeneration in Labrador retriever dogs showing clinical signs similar to human STGD. To investigate the genetic cause of the disease, we used whole-genome sequencing of a family quartet including two affected offspring and their unaffected parents. This led to the identification of a loss-of-function mutation in the ABCA4 gene. The findings of this study may enable the development of a canine model for human STGD.

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

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in humans and dogs. Currently, no standard treatment is available but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs1395) was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium. The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.

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. Developmental Dynamics 247:630–641, 2018.

Retinoblastoma, an intraocular pediatric cancer, develops in the embryonic retina following biallelic loss of RB1. However, there is a wide range of genetic and epigenetic changes that can affect RB1 resulting in different clinical outcomes. In addition, other transformations, such as MYCN amplification, generate particularly aggressive tumors, which may or may not be RB1 independent. Recognizing the cellular characteristics required for tumor development, by identifying the elusive cell‐of‐origin for retinoblastoma, would help us understand the development of these tumors. In this review we summarize the heterogeneity reported in retinoblastoma on a molecular, cellular and tissue level. We also discuss the challenging heterogeneity in current retinoblastoma models and suggest future platforms that could contribute to improved understanding of tumor initiation, progression and metastasis in retinoblastoma, which may ultimately lead to more patient‐specific treatments.

Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner. Barring patterns on individual feathers are widespread phenomena in a number of wild bird species. Still, the genetic background and molecular mechanisms that give rise to barring remains poorly understood. Sex-linked barring is a striping pattern present on individual feathers in domestic chickens, which can be utilized as a model species to gain an understanding of the underlying ‘mode of action’ of biological pattern formation. Our findings suggest that regulatory mutations in the tumor suppressor gene CDKN2A first resulted in a primitive barring pattern and that two missense mutations in the same gene occurred later and independently, causing the more distinct barring pattern of extant chicken breeds. A plausible mechanism is that the altered expression of CDKN2A causes melanocyte progenitor cells to prematurely stop dividing and instead differentiate into pigment-producing cells. The temporary lack of melanocytes expresses itself as a white bar until the progenitor cells are replenished and pigment is produced to form a pigmented bar. It is remarkable that a good proportion of the world-wide production of animal protein is based on chicken that are carrying a functionally important missense mutation in the CDKN2A tumor suppressor gene.