POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Mouse retinal development: Role of cell adhesion and mechanism of gene regulation

Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^

THE FUNCTIONAL ORGANIZATION OF SYNAPTIC VESICLE POOLS IN A RETINAL BIPOLAR NEURON

Ribbon synapses are found in sensory systems and are characterized by ‘ribbon-like’ organelles that tether synaptic vesicles. The synaptic ribbons co-localize with sites of calcium entry and vesicle fusion, forming ribbon-style active zones. The ability of ribbon synapses to maintain rapid and sustained neurotransmission is critical for vision, hearing and balance. At retinal ribbon synapses, three vesicle pools have been proposed. A rapid pool of vesicles that are docked at the plasma membrane, and whose fusion is limited only by calcium entry, a releasable pool of ATP-primed vesicles whose size also correlates with the number of ribbon-tethered vesicles, and a reserve pool of non-ribbon-tethered cytoplasmic vesicles. However evidence of vesicle fusion at sites away from ribbon-style active zones questions this organization. Another fundamental question underlying the mechanism of vesicle fusion at these synapses is the role of SNARE (Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor) proteins. Vesicles at conventional neurons undergo SNARE complex-mediated fusion. However a recent study has suggested that ribbon synapses involved in hearing can operate independently of neuronal SNAREs. We used the well-characterized goldfish bipolar neuron to investigate the organization of vesicle pools and the role of SNARE proteins at a retinal ribbon synapse. We blocked functional refilling of the releasable pool and then stimulated bipolar terminals with brief depolarizations that triggered the fusion of the rapid pool of vesicles. We found that the rapid pool draws vesicles from the releasable pool and that both pools undergo release at ribbon-style active zones. To assess the functional role of SNARE proteins at retinal ribbon synapses, we used peptides derived from SNARE proteins that compete with endogenous proteins for SNARE complex formation. The SNARE peptides blocked fusion of reserve vesicles but not vesicles in the rapid and releasable pools, possibly because both rapid and releasable vesicles were associated with preformed SNARE complexes. However, an activity-dependent block in refilling of the releasable pool was seen, suggesting that new SNARE complexes must be formed before vesicles can join a fusion-competent pool. Taken together, our results suggest that SNARE complex-mediated exocytosis of serially-organized vesicle pools at ribbon-style active zones is important in the neurotransmission of vision.

Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^

(Table 1) Insoluble aerosol concentrations and vertical fluxes at the eastern shelf of the Black Sea during breeze circulation in October 1987

Amounts of aerosols transported to the shelf surface were calculated on the basis of in situ measurements of concentrations of eolian matter (insoluble aerosol fraction) and vertical fluxes of settling dust in five areas of the Black Sea shelf from the Danube delta to the Inguri River mouth. More than 8.3 mln t of eolian matter are annually transported from the land over the shelf of the former USSR. At the same time more than 5.4 mln t are supplied to the northwestern shelf area, 1.7 mln t are supplied to the Crimean area, about 0.8 mln t are supplied to the Kerch-Taman' area, and about 0.45 mln t are supplied to the Caucasian area.

Deep thermohaline Circulation in the low-latitude Atlantic during the Last Glacial

Present-day low-latitude eastern and western Atlantic basins are geochemically distinct below the sill depth of the Mid-Atlantic Ridge. While Antarctic Bottom Water (AABW) circulates freely in the western Atlantic, flow into the eastern Atlantic is restricted below 4 km which results in filling the abyssal depths of this basin with water of geochemical similarity to nutrient depleted North Atlantic Deep Water. Using carbon isotopes and Cd/Ca ratios in benthic foraminifera we reconstruct the geochemistry of these basins during the last glacial maximum. Results indicate that deep eastern and western Atlantic basins became geochemically identical during the last glacial. This was achieved by shoaling of the upper surface of AABW above the sill depth of the Mid-Atlantic Ridge, which allowed bottom waters in both basins to be filled with the same water mass. Although AABW became the dominant water mass in the deep eastern Atlantic basin during the glacial, Holocene-glacial delta13C-PO4 shifts in this basin are in Redfield proportions, unlike the disproportionate Holocene-glacial delta13C-PO4 shifts observed in the Southern Ocean. By examining the composition of deep and intermediate waters throughout the Atlantic, we show that this effect was induced by a change in gradient of the delta13C-PO4 deepwater mixing line during glacial times. Evidence from high-latitude planktonic data suggests that the change in gradient of the deepwater mixing line was brought about through a significant reduction in the thermodynamic effect on Southern Ocean surface waters. By using coupled delta13C-PO4 data to constrain the composition of end member water masses in the glacial Atlantic, we conclude that deep waters in the low-latitude glacial Atlantic were composed of a mixture of northern and southern source waters in a ratio of 1:3.

Carbonate cements and fluid circulation of ODP Leg 116 samples

The carbonate cements found in Sites 717-719 of ODP Leg 116 correspond to the precipitation of inorganic calcite due to circulation of hot fluid associated with intraplate deformation in the central Indian Ocean. A first burst of hydrothermal activity may have occurred 7.5-9 Ma and a second burst less than 0.5 Ma. These fluids were probably derived from the basaltic basement and the immediately overlying sediments.

Benthic foraminiferal carbon isotopic records and the development of abyssal circulation at DSDP Sites 9-77, 12-119, 30-289, 82-563 and 94-608

The North Atlantic at present is ventilated by overflow of the Denmark Strait, Iceland-Faeroe Ridge, Faeroe Bank Channel, and Wyville-Thompson Ridge. The evolution of Cenozoic abyssal circulation of this region was related to tectonic opening and subsidence of these sills. We used d13C records of the benthic foraminifer Cibicidoides to decipher the timing of tectonically controlled changes in bottom-water circulation in the eastern basins (Biscay and Iberian) of the northern North Atlantic. Records from Site 608 (Kings Trough, northeastern North Atlantic) show that from about 24 to 15 Ma (early to early middle Miocene), d13C values in the Kings Trough area were depleted relative to western North Atlantic values and were more similar to Pacific d13C values. This reflects less ventilation of the Kings Trough region as compared to the well-oxygenated western North Atlantic. Comparison of Oligocene d13C records from Site 119 (Bay of Biscay) with western North Atlantic records suggests that the eastern basin was also relatively isolated prior to 24 Ma. At about 15 Ma, d13C values at Site 608 attained values similar to the western North Atlantic, indicating increased eastern basin ventilation in the middle Miocene. This increased advection into the eastern basin predated a major d18O increase which occurred at about 14.6 Ma. Subsidence estimates of the Greenland-Scotland Ridge indicate that the deepening of the Iceland-Faeroe Ridge was coincident with the marked change in eastern basin deep-water ventilation.

Past circulation along the western Iberian margin

Fifteen Iberian margin sediment cores, distributed between 43°12'N and 35°53'N, have been used to reconstruct spatial and temporal (sub)surface circulation along the Iberian margin since the Last Glacial period. Time-slice maps of planktonic foraminiferal derived summer sea surface temperature (SST) and export productivity (Pexp) were established for specific time intervals within the last 35 ky: the Holocene (Recent and last 8 ky), Younger Dryas (YD), Heinrich Stadials (HS) 1, 2a, 2b, 3, and the Last Glacial Maximum (LGM). The SST during the Holocene shows the same latitudinal gradient along the western Iberian margin as present-day with cold but productive areas that reflect the influence of coastal upwelling centers. The LGM appears as a slightly less warm, but more productive period relative to the Holocene and present-day conditions, suggesting that sea-level minima forced a westward displacement of the coastal upwelling centers possibly accompanied by a strengthening of northward winds. During the YD, a longitudinal thermal front is depicted at 10°W, with cold polar waters offshore and warmer subtropical waters inshore, suggesting that the subtropical Paleo-Iberian Poleward Current more likely flowed at a more inshore location masking the local SST signal and amplitude of variation. A substantial cooling and drop in productivity is observed during all HS, in particular HS1 and HS3, reflecting the penetration of icebergs-derived meltwater. These most extreme southward extensions of very cold waters define a strong SST gradient that marks a possible Paleo-Azores Front. Higher production south of this front was likely fed by frontal nutrient advection.

Isopollen maps for 18,000 years B.P. of the offshore of Northwest Africa: Evidence for paleowind circulation

Distribution patterns of the most important pollen types from southern European and northwest African source areas for the 18,000 years B.P. time slice are reconstructed from pollen records of 14 well-dated deep-sea cores located between 37° and 9°N and compared with the modern pollen distribution in this area. It is concluded that the belt with maximum African Easterly Jet transport did not shift latitudinally during the last glacial-interglacial transition but remained at about 20°N. Furthermore, it is substantiated that the trade winds did not shift latitudinally during the last glacial-interglacial transition. This evidence is not compatible with an atmospheric circulation model that assumes a zone of surface westerlies in the northern part of northwest Africa. Trade winds during glacial episodes did, however, intensify, especially from about 36° to 24° N.

On the effects of circulation, sediment resuspension and biological incorporation by diatoms in an ocean model of aluminium, link to model data

The distribution of dissolved aluminium in the West Atlantic Ocean shows a mirror image with that of dissolved silicic acid, hinting at intricate interactions between the ocean cycling of Al and Si. The marine biogeochemistry of Al is of interest because of its potential impact on diatom opal remineralisation, hence Si availability. Furthermore, the dissolved Al concentration at the surface ocean has been used as a tracer for dust input, dust being the most important source of the bio-essential trace element iron to the ocean. Previously, the dissolved concentration of Al was simulated reasonably well with only a dust source, and scavenging by adsorption on settling biogenic debris as the only removal process. Here we explore the impacts of (i) a sediment source of Al in the Northern Hemisphere (especially north of ~ 40° N), (ii) the imposed velocity field, and (iii) biological incorporation of Al on the modelled Al distribution in the ocean. The sediment source clearly improves the model results, and using a different velocity field shows the importance of advection on the simulated Al distribution. Biological incorporation appears to be a potentially important removal process. However, conclusive independent data to constrain the Al / Si incorporation ratio by growing diatoms are missing. Therefore, this study does not provide a definitive answer to the question of the relative importance of Al removal by incorporation compared to removal by adsorptive scavenging.

Circulation, eddies, oxygen and nutrient changes in the eastern tropical South Pacific Ocean

A large, subsurface oxygen deficiency zone is located in the eastern tropical South Pacific Ocean (ETSP). The large-scale circulation in the eastern equatorial Pacific and off Peru in November/December 2012 shows the influence of the equatorial current system, the eastern boundary currents, and the northern reaches of the subtropical gyre. In November 2012 the Equatorial Undercurrent is centered at 250 m depth, deeper than in earlier observations. In December 2012 the equatorial water is transported southeastward near the shelf in the Peru-Chile Undercurrent with a mean transport of 1.6 Sv. In the oxygen minimum zone (OMZ) the flow is overlaid with strong eddy activity on the poleward side of the OMZ. Floats with parking depth at 400 m show fast westward flow in the mid-depth equatorial channel and sluggish flow in the OMZ. Floats with oxygen sensors clearly show the passage of eddies with oxygen anomalies. The long-term float observations in the upper ocean lead to a net community production estimate at about 18° S of up to 16.7 mmol C m?3 yr1 extrapolated to an annual rate and 7.7 mmol C m?3 yr?1 for the time period below the mixed layer. Oxygen differences between repeated ship sections are influenced by the Interdecadal Pacific Oscillation, by the phase of El Niño, by seasonal changes, and by eddies and hence have to be interpreted with care. At and south of the equator the decrease in oxygen in the upper ocean since 1976 is related to an increase in nitrate, phosphate, and in part in silicate.

Multimodal Imaging of Cotton Wool Spots in Branch Retinal Vein Occlusion.

PURPOSE: To describe and follow cotton wool spots (CWS) in branch retinal vein occlusion (BRVO) using multimodal imaging. METHODS: In this prospective cohort study including 24 patients with new-onset BRVO, CWS were described and analyzed in color fundus photography (CF), spectral domain optical coherence tomography (SD-OCT), infrared (IR) and fluorescein angiography (FA) every 3 months for 3 years. The CWS area on SD-OCT and CF was evaluated using OCT-Tool-Kit software: CWS were marked in each single OCT B-scan and the software calculated the area by interpolation. RESULTS: 29 central CWS lesions were found. 100% of these CWS were visible on SD-OCT, 100% on FA and 86.2% on IR imaging, but only 65.5% on CF imaging. CWS were visible for 12.4 ± 7.5 months on SD-OCT, for 4.4 ± 3 months and 4.3 ± 3.4 months on CF and on IR, respectively, and for 17.5 ± 7.1 months on FA. The evaluated CWS area on SD-OCT was larger than on CF (0.26 ± 0.17 mm(2) vs. 0.13 ± 0.1 mm(2), p < 0.0001). The CWS area on SD-OCT and surrounding pathology such as intraretinal cysts, avascular zones and intraretinal hemorrhage were predictive for how long CWS remained visible (r(2) = 0.497, p < 0.002). CONCLUSIONS: The lifetime and presentation of CWS in BRVO seem comparable to other diseases. SD-OCT shows a higher sensitivity for detecting CWS compared to CF. The duration of visibility of CWS varies among different image modalities and depends on the surrounding pathology and the CWS size.