Sonic hedgehog promotes rod photoreceptor differentiation in mammalian retinal cells in vitro.

Journal ArticleThe hedgehog gene family encodes secreted proteins important in many developmental patterning events in both vertebrates and invertebrates. In the Drosophila eye disk, hedgehog controls the progression of photoreceptor differentiation in the morphogenetic furrow. To investigate whether hedgehog proteins are also involved in the development of the vertebrate retina at stages of photoreceptor differentiation, we analyzed expression of the three known vertebrate hedgehog genes. We found that Sonic hedgehog and Desert hedgehog are expressed in the developing retina, albeit at very low levels, whereas Indian hedgehog (Ihh) is expressed in the developing and mature retinal pigmented epithelium, beginning at embryonic day 13. To determine whether hedgehog proteins have activities on developing retinal cells, we used an in vitro system in which much of retinal histogenesis is recapitulated. N-terminal recombinant Sonic Hedgehog protein (SHH-N) was added to rat retinal cultures for 3-12 d, and the numbers of retinal cells of various phenotypes were analyzed by immunohistochemistry. We found that SHH-N caused a transient increase in the number of retinal progenitor cells, and a 2- to 10-fold increase in the number of photoreceptors differentiating in the cultures when analyzed with three different photoreceptor-specific antigens. In contrast, the numbers of retinal ganglion cells and amacrine cells were similar to those in control cultures. These results show that Hedgehog proteins can regulate mitogenesis and photoreceptor differentiation in the vertebrate retina, and Ihh is a candidate factor from the pigmented epithelium to promote retinal progenitor proliferation and photoreceptor differentiation

Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells

<p>Abstract</p> <p>Background</p> <p>Maintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (<it>Ccnd1</it>^-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.</p> <p>Results</p> <p>In this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.</p> <p>Conclusion</p> <p>Our findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic <it>Ccnd1</it>^-/-retina.</p

X-ray Nova XTE J1550-564: RXTE Spectral Observations

Excellent coverage of the 1998 outburst of the X-ray Nova XTE J1550-564 was provided by the Rossi X-ray Timing Explorer. XTE J1550-564 exhibited an intense (6.8 Crab) flare on 1998 September 19 (UT), making it the brightest new X-ray source observed with RXTE. We present a spectral analysis utilizing 60 Proportional Counter Array spectra from 2.5-20 keV spanning 71 days, and a nearly continuous All Sky Monitor light curve. The spectra were fit to a model including multicolor blackbody disk and power-law components. XTE J1550-564 is observed in the very high, high/soft, and intermediate canonical outburst states of Black Hole X-ray Novae.Comment: 14 pages including 1 table and 4 figures, Accepted to ApJ Letter

Complete RXTE Spectral Observations of the Black Hole X-ray Nova XTE J1550-564

We report on the X-ray spectral behavior of the exceptionally bright X-ray nova XTE J1550-564 during its 1998-99 outburst. Our study is based on 209 pointed observations using the PCA and HEXTE instruments onboard the Rossi X-ray Timing Explorer spanning 250 days and covering the entire double-peaked eruption that occurred from 1998 September until 1999 May. The spectra are fit to a model including multicolor blackbody disk and power-law components. The source is observed in the very high and high/soft outburst states of black hole X-ray novae. During the very high state, when the power-law component dominated the spectrum, the inner disk radius is observed to vary by more than an order of magnitude; the radius decreased by a factor of 16 in one day during a 6.8 Crab flare. If the larger of these observed radii is taken to be the last stable orbit, then the smaller observed radius would imply that the inner edge of the disk is inside the event horizon! However, we conclude that the apparent variations of the inner disk radius observed during periods of increased power-law emission are probably caused by the failure of the multicolor disk/power-law model; the actual physical radius of the inner disk may remain fairly constant. This interpretation is supported by the fact that the observed inner disk radius remains approximately constant over 120 days in the high state, when the power-law component is weak, even though the disk flux and total flux vary by an order of magnitude. The mass of the black hole inferred by equating the approximately constant inner disk radius observed in the high/soft state with the last stable orbit for a Schwarzschild black hole is M_BH = 7.4 M_sun (D/6 kpc) (cos i)^{-1/2}.Comment: Submitted to ApJ, 20 pages including 6 figures + 4 large table

MURIN: Multimodal Retinal Imaging and Navigated-laser-delivery for dynamic and longitudinal tracking of photodamage in murine models

IntroductionLaser-induced photodamage is a robust method for investigating retinal pathologies in small animals. However, aiming of the photocoagulation laser is often limited by manual alignment and lacks real-time feedback on lesion location and severity. Here, we demonstrate MURIN: MUltimodal Retinal Imaging and Navigated-laser-delivery, a multimodality OCT and SLO ophthalmic imaging system with an image-guided scanning laser lesioning module optimized for the murine retina. The proposed system enables targeting of focal and extended area lesions under OCT guidance to benefit visualization of photodamage response and the precision and repeatability of laser lesion models of retinal injury.MethodsMURIN optics were optimized for simultaneous near-infrared and visible wavelength imaging/laser lesioning. Custom LabView control software was developed to steer the photocoagulation laser and automatically deliver laser pulses to targets-of-interest. In vivo retinal imaging was performed in transgenic Müller glia-tdTomato reporter mice (Rlbp1:CreER; Rosaai14, 5 animals, 10 eyes) and microglia-GFP/Müller glia-tdTomato reporter mice (Cx3cr1GFP; Rlbp1:CreER; Rosaai14, 9 animals, 15 eyes) to visualize cellular changes in the retina after laser lesion delivery.ResultsReal-time MURIN imaging concurrent with laser lesioning allowed us to visualize lesion formation dynamics and any corresponding changes in retinal morphology. We observe increasing fluorescence photoconversion on SLO and scattering contrast on OCT. Significant morphological changes are visible on MURIN after high-severity photodamage. OCT cross-sections show the spatial extent of the lesions contract over time from diffusion areas of increased scattering to granular scatterers and corresponding SLO images show a radial pattern surrounding severe focal lesions, which may be a result of a change in Müller cell shape or orientation in response to injury. The inner plexiform layer is distorted and increased RPE thickness and scattering are observed, all of which are confirmed on corresponding hematoxylin and eosin (H&amp;E) histology and differential interference contrast (DIC) microscopy.DiscussionMURIN as a unique imaging platform that enables combined SLO and OCT imaging with an integrated image-guided laser lesioning module. This technology has clear benefits over existing multimodal imaging and laser lesioning systems by enabling simultaneous multimodal imaging, independent and precise control of Iridex laser pulse parameters and patterns, and real-time OCT and SLO visualization of lesion formation

Proliferative reactive gliosis is compatible with glial metabolic support and neuronal function

<p>Abstract</p> <p>Background</p> <p>The response of mammalian glial cells to chronic degeneration and trauma is hypothesized to be incompatible with support of neuronal function in the central nervous system (CNS) and retina. To test this hypothesis, we developed an inducible model of proliferative reactive gliosis in the absence of degenerative stimuli by genetically inactivating the cyclin-dependent kinase inhibitor <it>p27^Kip1</it>(<it>p27 </it>or <it>Cdkn1b</it>) in the adult mouse and determined the outcome on retinal structure and function.</p> <p>Results</p> <p>p27-deficient Müller glia reentered the cell cycle, underwent aberrant migration, and enhanced their expression of intermediate filament proteins, all of which are characteristics of Müller glia in a reactive state. Surprisingly, neuroglial interactions, retinal electrophysiology, and visual acuity were normal.</p> <p>Conclusion</p> <p>The benign outcome of proliferative reactive Müller gliosis suggests that reactive glia display context-dependent, graded and dynamic phenotypes and that reactivity in itself is not necessarily detrimental to neuronal function.</p

A surface groove essential for viral Bcl-2 function during chronic infection in vivo

Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the gamma-herpesvirus 68 (gammaHV68) Bcl-2 family protein (gammaHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the gammaHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type gammaHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of gammaHV68 from latency and efficient persistent gammaHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection