Marek Pacal

Noninvasive, In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography

Background Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration. Methodology/Principal Findings We achieved to adapt a commercial 3rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Lebers congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified. Conclusions/Significance We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.

Division and apoptosis of E2f-deficient retinal progenitors

The activating E2f transcription factors (E2f1, E2f2 and E2f3) induce transcription and are widely viewed as essential positive cell cycle regulators. Indeed, they drive cells out of quiescence, and the ‘cancer cell cycle’ in Rb1 null cells is E2f-dependent. Absence of activating E2fs in flies or mammalian fibroblasts causes cell cycle arrest, but this block is alleviated by removing repressive E2f or the tumour suppressor p53, respectively. Thus, whether activating E2fs are indispensable for normal division is an area of debate. Activating E2fs are also well known pro-apoptotic factors, providing a defence against oncogenesis, yet E2f1 can limit irradiation-induced apoptosis. In flies this occurs through repression of hid (also called Wrinkled; Smac/Diablo in mammals). However, in mammals the mechanism is unclear because Smac/Diablo is induced, not repressed, by E2f1, and in keratinocytes survival is promoted indirectly through induction of DNA repair targets. Thus, a direct pro-survival function for E2f1–3 and/or its relevance beyond irradiation has not been established. To address E2f1–3 function in normal cells in vivo we focused on the mouse retina, which is a relatively simple central nervous system component that can be manipulated genetically without compromising viability and has provided considerable insight into development and cancer. Here we show that unlike fibroblasts, E2f1–3 null retinal progenitor cells or activated Müller glia can divide. We attribute this effect to functional interchangeability with Mycn. However, loss of activating E2fs caused downregulation of the p53 deacetylase Sirt1, p53 hyperacetylation and elevated apoptosis, establishing a novel E2f–Sirt1–p53 survival axis in vivo. Thus, activating E2fs are not universally required for normal mammalian cell division, but have an unexpected pro-survival role in development.

Insights from animal models on the origins and progression of retinoblastoma.

The RB gene was discovered 20 years ago because of its role in the childhood eye cancer retinoblastoma. However, surprisingly little progress was made in defining the role of RB protein in the retina. In the last two years, new models exploiting conditional deletion of the mouse Rb gene have altered this picture radically. These models provide insight into the first Rb function, the cell of origin of retinoblastoma, the window during which Rb acts, distinct cell-specific defenses against Rb loss, the number and type of post-Rb lesions required for transformation, why pediatric tumors exist, the controversial role of the p53 pathway in retinoblastoma, and the reason why the disease is virtually unique to humans. Two years have dramatically improved our understanding of Rb function in the tissue that gave us this important tumor suppressor.

Direct and indirect effects of hedgehog pathway activation in the mammalian retina

The morphogen Sonic hedgehog (Shh) is expressed by the projection neurons of the retina, retinal ganglion cells (RGCs) and promotes retinal precursor cell (RPC) proliferation. To distinguish between direct and indirect effects of Hedgehog (Hh) pathway activation in the perinatal mouse retina, we followed the fate of cells that expressed a constitutively active allele of Smoothened (SMO-M2), the signal transduction component of the Hh pathway. SMO-M2 expression promoted a cell-autonomous increase in CyclinD1 expression and RPC proliferation and promoted the development of cells with an inner nuclear layer identity. SMO-M2 expression also inhibited rhodopsin expression in uninfected cells, thus highlighting an unexpected non-cell autonomous effect of Hh pathway activation on photoreceptor development.

The RB protein family in retinal development and retinoblastoma: new insights from new mouse models.

The Rb gene was isolated almost 20 years ago, but fundamental questions regarding its role in retinal development and retinoblastoma remain. What is the normal function of RB protein in retinogenesis? What is the cell-of-origin of retinoblastoma? Why do retinoblastoma tumors have recurrent genetic lesions other than Rb inactivation? Why is retinoblastoma not induced by defects in cell cycle regulators other than Rb? Why is the retina so sensitive to Rb loss? Recently developed conditional Rb knockout models provide new insight into some of these issues. The data suggest that RB protein may not control the rate of progenitor division, but is critical for cell cycle exit when dividing retinal progenitors differentiate into postmitotic transition cells. This finding focuses attention on the ectopically dividing transition cell, rather than the progenitor, as the cell-of-origin. Cell-specific analyses in the RB-deficient retina reveal that ectopically dividing photoreceptors, bipolar and ganglion cells die, but amacrine, horizontal and Müller cells survive and stop dividing when they terminally differentiate. Rare amacrine transition cells escape cell cycle exit and generate tumors. These data suggest that post-Rb mutations are required to overcome growth arrest associated with terminal differentiation, rather than apoptosis as previously suggested. To explain why perturbing cell cycle regulators other than RB does not initiate retinoblastoma, we speculate that mutations in other components of the RB pathway perturb cell cycle arrest, but only RB loss triggers genome instability in retinal transition cells, which may be critical to facilitate post-Rb mutations necessary for transformation. Cell-specific differences in the effect of Rb loss on genome stability may contribute to the tremendous sensitivity of retinal transition cells to tumorigenesis. The new mouse models of retinoblastoma will be invaluable for testing these possibilities.

Mapping differentiation kinetics in the mouse retina reveals an extensive period of cell cycle protein expression in post-mitotic newborn neurons

Background: Knowledge of gene expression kinetics around neuronal cell birth is required to dissect mechanisms underlying progenitor fate. Here, we timed cell cycle and neuronal protein silencing/induction during cell birth in the developing murine retina. Results: The pan‐cell cycle markers Pcna and Mcm6 were present in the post‐mitotic ganglion cell layer. Although confined to the neuroblastic layer (NBL), 6–7% of Ki67+ cells lacked six progenitor/cell cycle markers, and expressed neuronal markers. To define protein extinction/induction timing, we defined G2/M length throughout retinogenesis, which was typically 1–2 h, but <10% cells took double this time. BrdU‐chase analyses revealed that at E12.5, Tubb3 (Tuj1) appeared at M‐phase, followed by Calb2 and Dcx at ∼2 h, Elavl2/3/4 at ∼4 h, and Map2 at ∼6 h after cell birth, and these times extended with embryonic age. Strikingly, Ki67 was not extinguished until up to a day after cell cycle exit, coinciding with exit from the NBL and induction of late markers such as Map1b/Uchl1/Rbfox3. Conclusions: A minor population of progenitors transits slowly through G2/M and, most importantly, some cell cycle proteins are retained for an unexpectedly long period in post‐mitotic neurons. The high‐resolution map of cell birth kinetics reported here provides a framework to better define mechanisms that regulate neurogenesis. Developmental Dynamics 241:1525–1544, 2012.

A CDK2 activity signature predicts outcome in CDK2-low cancers

The role of cyclin-dependent kinase 2 (CDK2) in cancer is controversial. A major hurdle is the availability of tools to easily assess its activity across many samples. Here, we introduce a transcriptional signature to specifically track CDK2 activity. It responds to genetic and chemical perturbations in the CDK-RB-E2F axis, correlates with mitotic rate in vitro and in vivo and reacts rapidly to changes in CDK2 activity during cell cycle progression. We find that CDK2 activity is specifically elevated in human testes, mirroring its critical function in mice, and report very distinct profiles across human cancers. Increased CDK2 activity decreases risk in colon cancer, but elevates poor outcome two- to fivefold in specific tumors, including low grade glioma, kidney, thyroid, adrenocortical and prostate cancer. These are typically ‘CDK2-low’ cancers, suggesting that above a certain threshold CDK2 promotes progression, but further increases do not influence outcome. Multivariate analysis revealed that the CDK2 signature is the most important predictive feature in these cancers versus dozens of other clinical parameters, such as tumor grade or mitotic index. Thus, transcriptome data provides a novel, straightforward method to monitor CDK2 activity, implicates key roles for the kinase in a subset of human tissues and tumors and enhances cancer risk prediction. The strategy used here for CDK2 could be applied to other kinases that influence transcription.

Abstract 1092: Alterations of the retinoblastoma gene in stage III breast cancers

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We report the novel finding of point mutations affecting the RB1 gene in breast cancer tissue. Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution; Leu607Ile, Arg698Trp, and Arg621Cys, respectively. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring Leu607Ile. All three RB1 point mutations encoded stable nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Interestingly, Leu607Ile and Arg621Cys are both located within the spacer region of the protein. Mutations in this part of the RB1 gene have previously been identified in retinoblastoma-prone families. Multiple sequence alignment across species indicates this area to be evolutionary conserved. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046). Although rare, our findings suggest RB1 mutations to be of biological importance in breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1092.