Rb-Mediated Neuronal Differentiation through Cell-Cycle–Independent Regulation of E2f3a

It has long been known that loss of the retinoblastoma protein (Rb) perturbs neural differentiation, but the underlying mechanism has never been solved. Rb absence impairs cell cycle exit and triggers death of some neurons, so differentiation defects may well be indirect. Indeed, we show that abnormalities in both differentiation and light-evoked electrophysiological responses in Rb-deficient retinal cells are rescued when ectopic division and apoptosis are blocked specifically by deleting E2f transcription factor (E2f) 1. However, comprehensive cell-type analysis of the rescued double-null retina exposed cell-cycle–independent differentiation defects specifically in starburst amacrine cells (SACs), cholinergic interneurons critical in direction selectivity and developmentally important rhythmic bursts. Typically, Rb is thought to block division by repressing E2fs, but to promote differentiation by potentiating tissue-specific factors. Remarkably, however, Rb promotes SAC differentiation by inhibiting E2f3 activity. Two E2f3 isoforms exist, and we find both in the developing retina, although intriguingly they show distinct subcellular distribution. E2f3b is thought to mediate Rb function in quiescent cells. However, in what is to our knowledge the first work to dissect E2f isoform function in vivo we show that Rb promotes SAC differentiation through E2f3a. These data reveal a mechanism through which Rb regulates neural differentiation directly, and, unexpectedly, it involves inhibition of E2f3a, not potentiation of tissue-specific factors

Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic β-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity

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 3(rd) 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 Leber's 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

Coordinating Cell Cycle Exit and Differentiation in the Mammalian Retina and its Dependence on Rb

Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either side of mitosis. Instead of mitosis behaving as a cog that activates post-exit differentation events we suggest that a common trigger induces both the exit and differentiation programs in RPCs, precisely coordinating their startpoints, but that each subsequent cascade unfolds independently. This model explains the convergence of birth and differentiation but also their temporal maliability. This view fits with our observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin.Ph

Capability of the OCT to detect and capture the nature of lesions.

<p>A)–F) Multiple retinal rosette formation in the neural retinal leucine zipper (<i>Nrl</i>) knockout mouse. A) SLO surface image (514 nm) in which the retinal rosettes (arrow) show as whitish dots. B) SLO autofluorescence image indicating that the rosettes contain fluorescent material. C) Representative OCT slice of a <i>Nrl</i> knockout mouse revealing details of the nature of the rosettes (arrow) and their depth localization. D) Comparison of the OCT representation of <i>Nrl</i> rosettes with histology (different individual animal). E, F) Detail illustrating how well retinal structures in OCT and histology correlate.</p

Topographic analysis of retinal thickness in an organ-specific model of retinoblastoma protein (<i>Rb</i>) deficiency.

<p>Thickness variations (center vs. periphery) were caused by imperfections of the Cre-lox system (see text), leading to differences in developmental apoptosis. A) Histological section across the central retina showing the smooth transition between centrally normal and peripherally reduced thickness. B) OCT section of the same region, the retinal thickness correlating well with the histomorphological data. C) Assessment of the gradual changes of retinal thickness from center to (mid)periphery based on 5 manually placed OCT slices. Left: SLO image of the fundus region with the position of the slices superimposed. Right: OCT slices at the positions indicated, ordered from center to periphery. D) Topography of retinal thickness calculated from 92 equidistant OCT slices (“volume scan” data). The color scale values are in µm.</p

Capability of the OCT to detect and capture the nature of lesions.

<p>A)–D) Site of neovascularization in a Crumbs 1 (<i>Crb1</i>) knockout mouse. A) SLO fluorescence angiographic (FLA) image of a retinal neovascular site (arrow) in a representative <i>Crb1</i> knockout mouse. B) Detail of g) illustrating the traction the aberrant vessel applies to the neighboring capillaries. C) Representative OCT slice depicting enlarged aberrant retinal vessels (black arrow) as well as choroidal vascular changes (white arrow) at the same position, implicating a connection between both vascular beds. D) Histological section of the above neovascular site for comparison. The black arrow points to aberrant retinal vessels, and the white arrow to choroidal changes.</p

OCT assessment of light-induced murine retinal degeneration.

<p>A) OCT section across the central retina, containing adjacent damaged and non-damaged areas. B) Demarcation of the damaged area <i>in vivo</i> by SLO autofluorescence (AF) imaging based on fluorescent photoreceptor debris (marked by an asterisk). C) Detail of the transition zone between damaged and non-damaged retina in a). The asterisk marks the damaged area as in B). D), E) Comparison of the representation of light-induced retinal damage in histology and OCT. The arrowhead in the OCT image points towards a site of retinal edema.</p

OCT and histological morphometric data in rodents.

<p>Relationship between OCT and histological morphometry in C57/BL6 mice broken down to retinal layers. A) Correlation of histological and OCT data. Pearson's correlation coefficient (R²) is based on the data of all quantified retinal layers. B) Comparison of retinal layer thickness between histology (dark) and OCT (light). There was no statistically significant difference between histological analysis and OCT-based quantification in any retinal layer using Student's t-test at a significance level of p<0.05. All data are reported as mean values±standard deviation (error bars).</p

Principle of Optical Coherence Tomography (OCT) and its application in rodents.

<p>A) Schematic diagram of spectral-domain (SD) OCT. Green arrows indicate efferent, red arrows afferent light. B) Comparison of OCT to conventional ophthalmic imaging techniques. Top left: Conventional techniques either yield surface images of the retina (“fundus”), or in case of Scanning-Laser Ophthalmoscopy (SLO), confocal horizontal sections. Top right: Example of an SLO image of the central murine retina. Bottom left: In contrast, the OCT provides high-resolution vertical sections. Bottom right: Example of an OCT slice from the central murine retina in comparison to matching standard light microscopy. C) Representation of retinal layers in OCT and histology. See text for details. D) OCT recording setup for rodents. The schematic drawing of a mouse marks the recording position on the XYZ table. The eye is directly facing the OCT recording unit with a 78 dpt. lens attached.</p