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The large DMD gene encodes a group of dystrophin proteins in brain and retina, produced from multiple promoters and alternative splicing events. Dystrophins are core components of different scaffolding complexes in distinct cell types. Their absence may thus alter several cellular pathways, which might explain the heterogeneous genotype–phenotype relationships underlying central comorbidities in Duchenne muscular dystrophy (DMD). However, the cell-specific expression of dystrophins and associated proteins (DAPs) is still largely unknown. The present study provides a first RNA-Seq-based reference showing tissue- and cell-specific differential expression of dystrophins, splice variants and DAPs in mouse brain and retina. We report that a cell type may express several dystrophin complexes, perhaps due to expression in separate cell subdomains and/or subpopulations, some of which with differential expression at different maturation stages. We also identified new splicing events in addition to the common exon-skipping events. These include a new exon within intron 51 (E51b) in frame with the flanking exons in retina, as well as inclusions of intronic sequences with stop codons leading to the presence of transcripts with elongated exons 40 and/or 41 (E40e, E41e) in both retina and brain. PCR validations revealed that the new exons may affect several dystrophins. Moreover, immunoblot experiments using a combination of specific antibodies and dystrophin-deficient mice unveiled that the transcripts with stop codons are translated into truncated proteins lacking their C-terminus, which we called N-Dp427 and N-Dp260. This study thus uncovers a range of new findings underlying the complex neurobiology of DMD.

16 p.-7 fig., Cone photoreceptor cell death in inherited retinal diseases, such as Retinitis Pigmentosa (RP), leads to the loss of high acuity and color vision and, ultimately to blindness. In RP, a vast number of mutations perturb the structure and function of rod photoreceptors, while cones remain initially unaffected. Extensive rod loss in advanced stages of the disease triggers cone death by a mechanism that is still largely unknown. Here, we show that secondary cone cell death in animal models for RP is associated with increased activity of histone deacetylates (HDACs). A single intravitreal injection of an HDAC inhibitor at late stages of the disease, when the majority of rods have already degenerated, was sufficient to delay cone death and support long-term cone survival in two mouse models for RP, affected by mutations in the phosphodiesterase 6b gene. Moreover, the surviving cones remained light-sensitive, leading to an improvement in visual function. RNA-seq analysis of protected cones demonstrated that HDAC inhibition initiated multi-level protection via regulation of different pro-survival pathways,including MAPK, PI3K-Akt, and autophagy. This study suggests a unique opportunity for targeted pharmacological protection of secondary dying cones by HDAC inhibition and creates hope to maintain vision in RP patients even in advanced disease stages., This work was supported by the ProRetina foundation,the Kerstan Foundation, Deutsche Forschungsgemeinschaft (DFGTR 1238/4-1), Swiss National Science Foundation (31003A_173008),BMBF (FKZ: 01EK1613E), and GC2018-098557-B-I00 from MCIU/AEI/FEDER, UE. RGS is a recipient of a JIN grant RTI2018-098990- J-I00 from MCIU, Spain.

Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): DESKi Background Left ventricular ejection fraction (EF) is a key parameter to assess cardiovascular functions in 2D cardiac ultrasound, but its manual assessment is time-consuming and subject to high inter and intra-observer variability. Deep learning-based approaches have been proposed to automatically predict EF but can suffer from a lack of explainability and interpretability, which limit the trust of the clinician and prevent errors from being detected. Purpose In this context, we propose a fully automatic method to reliably evaluate the biplane left ventricular EF on 2D echocardiography while following the modified Simpson’s rule calculation steps to provide explicit details to clinicians. Methods A deep learning model based on U-Net architecture was trained on internal apical 4 and 2-chamber echocardiographic images to segment the left ventricle and locate the mitral valve. Predicted segmentations are then validated with a statistical shape model which detects potential failures that could impact the final EF evaluation. Finally, end-diastolic and end-systolic frames are identified thanks to a spline interpolated from the remaining LV segmentations’ areas, and the EF is estimated on all available cardiac cycles. This approach was trained on a dataset of 783 patients. Its performances were evaluated on an internal dataset of 200 patients with a large diversity of EF and on an external openly available dataset containing 450 patients. Results On the internal dataset, EF assessment achieved a mean absolute error of 6.10% and a bias of 1.56 ± 7.58% using multiple cardiac cycles and removing failed segmentations. Regarding end-diastolic and end-systolic volumes, the mean absolute error was evaluated at 13.75mL and 10.70mL respectively. Segmentation performances were evaluated with the Dice and the Hausdorff distance, respectively at 0.92 ± 0.41 and 6.13 ± 2.9mm. On the external dataset, the approach predicted EF with a mean absolute error of 5.39% and a bias of -0.74 ± 7.12%. The mean absolute error for end-diastolic volume was 15.40mL and for end-systolic volume 8.18mL. Conclusions Following the recommended guidelines, we proposed an end-to-end fully automatic approach achieving state-of-the-art performances in EF evaluation in 2D cardiac ultrasound while giving explicit details to the clinicians at each step of the assessment. Abstract Figure. End-to-end fully automatic approach

Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): DESKi Background Transthoracic echocardiography requires long and demanding training. The use of deep learning algorithms for the identification of diagnostic quality reference views opens the way for assisting low-experienced operators. However, such algorithms have only been trained and validated on retrospective data extracted from daily hospital practice. Those retrospective data are the best clips selected by experts during each exam. Thus, the performance of those algorithms has not been evaluated on complete acquisitions, including the search phase, i.e. when the operator is moving the probe to reach the optimal position. Those search phases are not recorded in clinical practice. Purpose The objective of this study was to evaluate the performance of a deep learning algorithm that identifies diagnostic quality images for 7 echocardiographic reference views on a prospective dataset including search phases. Methods A retrospective dataset containing acquisitions extracted from daily hospital practice was created. The retrospective dataset was manually annotated by experts following quality criteria that were defined for 7 main reference views: parasternal long and short axis, apical 4, 2, and 3-chamber, subcostal 4-chamber and inferior vena cava. Each frame of the acquisitions was annotated by an expert and reviewed by a second expert. A deep learning model was trained on the retrospective dataset and evaluated with 5-fold cross-validation. In a prospective dataset, for each included patient and each reference view, operators recorded the entire acquisition, including the search phase. Acquisitions were manually annotated by 2 experts following the same quality criteria. The model trained on retrospective data was evaluated on this prospective dataset against manual experts’ annotations. Additionally, 3 experts annotated a subset of the prospective dataset to evaluate the inter-observer variability. Results 481,111 frames from 1,325 patients were annotated for the retrospective dataset. On the 5-fold cross-validation, the algorithm reached an average accuracy of 89.2 ± 0.7% per frame and an average F1-score of 87.9 ± 0.9% per frame. For the prospective dataset, 70 patients were included and 143,804 frames annotated. The average accuracy was 84.8 ± 5.0% per patient, and the average F1-score was 83.1 ± 8.7% per patient. The average inter-observer agreement rates, performed on 10 patients (23,390 frames), were between 82.8 ± 4.9% and 86.3 ± 6.2%. Conclusions We propose a deep learning algorithm that automatically identifies 7 of the most common reference views with diagnostic quality while achieving performance within the range of the inter-observer variability in acquisitions including search phases.

Hippo signalling regulates eye growth during embryogenesis through its effectors YAP and TAZ. Taking advantage of a Yap heterozygous mouse line, we here sought to examine its function in adult neural retina, where YAP expression is restricted to Müller glia. We first discovered an unexpected temporal dynamic of gene compensation. At postnatal stages, Taz upregulation occurs, leading to a gain of function-like phenotype characterised by EGFR signalling potentiation and delayed cell-cycle exit of retinal progenitors. In contrast, Yap+/− adult retinas no longer exhibit TAZ-dependent dosage compensation. In this context, Yap haploinsufficiency in aged individuals results in Müller glia dysfunction, late-onset cone degeneration, and reduced cone-mediated visual response. Alteration of glial homeostasis and altered patterns of cone opsins were also observed in Müller cell-specific conditional Yap-knockout aged mice. Together, this study highlights a novel YAP function in Müller cells for the maintenance of retinal tissue homeostasis and the preservation of cone integrity. It also suggests that YAP haploinsufficiency should be considered and explored as a cause of cone dystrophies in human.

Mutations in the ubiquitously expressedpre-mRNA processing factor(PRPF)31gene, one of the most common causes of dominant form of Retinitis Pigmentosa (RP), lead to a retina-specific phenotype. It is uncertain which retinal cell types are affected and animal models do not clearly present the RP phenotype observed inPRPF31patients. Retinal organoids and retinal pigment epithelial (RPE) cells derived from human-induced pluripotent stem cells (iPSCs) provide potential opportunities for studying humanPRPF31-related RP. We demonstrate here that RPE cells carryingPRPF31mutations present important morphological and functional changes and thatPRPF31-mutated retinal organoids recapitulate the human RP phenotype, with a rod photoreceptor cell death followed by a loss of cones. The low level ofPRPF31expression may explain the defective phenotypes ofPRPF31-mutated RPE and photoreceptor cells, which were not observed in cells derived from asymptomatic patients or after correction of the pathogenic mutation by CRISPR/Cas9. Transcriptome profiles revealed differentially expressed and mis-spliced genes belonging to pathways in line with the observed defective phenotypes. The rescue of RPE and photoreceptor defective phenotypes byPRPF31gene augmentation provide the proof of concept for future therapeutic strategies.

BACKGROUND This study systematically investigated circulating and retinal tissue lipid determinants of human diabetic retinopathy (DR) to identify underlying lipid alterations associated with severity of DR. METHODS Retinal tissues were retrieved from postmortem human eyes, including 19 individuals without diabetes, 20 with diabetes but without DR, and 20 with diabetes and DR, for lipidomic study. In a parallel study, serum samples from 28 American Indians with type 2 diabetes from the Gila River Indian Community, including 12 without DR, 7 with mild nonproliferative DR (NPDR), and 9 with moderate NPDR, were selected. A mass-spectrometry–based lipidomic platform was used to measure serum and tissue lipids. RESULTS In the postmortem retinas, we found a graded decrease of long-chain acylcarnitines and longer-chain fatty acid ester of hydroxyl fatty acids, diacylglycerols, triacylglycerols, phosphatidylcholines, and ceramide(NS) in central retina from individuals with no diabetes to those with diabetes with DR. The American Indians’ sera also exhibited a graded decrease in circulating long-chain acylcarnitines and a graded increase in the intermediate-length saturated and monounsaturated triacylglycerols from no DR to moderate NPDR. CONCLUSION These findings suggest diminished synthesis of complex lipids and impaired mitochondrial β-oxidation of fatty acids in retinal DR, with parallel changes in circulating lipids. TRIAL REGISTRATION ClinicalTrials.gov NCT00340678. FUNDING This work was supported by NIH grants R24 DK082841, K08DK106523, R03DK121941, P30DK089503, P30DK081943, P30DK020572, P30 EY007003; The Thomas Beatson Foundation; and JDRF Center for Excellence (5-COE-2019-861-S-B).

International audience; The mdx52 mouse model of Duchenne muscular dystrophy (DMD) is lacking exon 52 of the DMD gene that is located in a hotspot mutation region causing cognitive deficits and retinal anomalies in DMD patients. This deletion leads to the loss of the dystrophin proteins, Dp427, Dp260 and Dp140, while Dp71 is preserved. The flash electroretinogram (ERG) in mdx52 mice was previously characterized by delayed dark-adapted b-waves. A detailed description of functional ERG changes and visual performances in mdx52 mice is, however, lacking. Here an extensive full-field ERG repertoire was applied in mdx52 mice and WT littermates to analyze retinal physiology in scotopic, mesopic and photopic conditions in response to flash, sawtooth and/or sinusoidal stimuli. Behavioral contrast sensitivity was assessed using quantitative optomotor response (OMR) to sinusoidally modulated luminance gratings at 100% or 50% contrast. The mdx52 mice exhibited reduced amplitudes and delayed implicit times in dark-adapted ERG flash responses, particularly in their b-wave and oscillatory potentials, and diminished amplitudes of light-adapted flash ERGs. ERG responses to sawtooth stimuli were also diminished and delayed for both mesopic and photopic conditions in mdx52 mice and the first harmonic amplitudes to photopic sine-wave stimuli were smaller at all temporal frequencies. OMR indices were comparable between genotypes at 100% contrast but significantly reduced in mdx52 mice at 50% contrast. The complex ERG alterations and disturbed contrast vision in mdx52 mice include features observed in DMD patients and suggest altered photoreceptor-to-bipolar cell transmission possibly affecting contrast sensitivity. The mdx52 mouse is a relevant model to appraise the roles of retinal dystrophins and for preclinical studies related to DMD.

Muller glial cells (MGCs) are responsible for the homeostatic and metabolic support of the retina. Despite the importance of MGCs in retinal disorders, reliable and accessible human cell sources to be used to model MGC‐associated diseases are lacking. Although primary human MGCs (pMGCs) can be purified from post‐mortem retinal tissues, the donor scarcity limits their use. To overcome this problem, we developed a protocol to generate and bank human induced pluripotent stem cell‐derived MGCs (hiMGCs). Using a transcriptome analysis, we showed that the three genetically independent hiMGCs generated were homogeneous and showed phenotypic characteristics and transcriptomic profile of pMGCs. These cells expressed key MGC markers, including Vimentin, CLU, DKK3, SOX9, SOX2, S100A16, ITGB1, and CD44 and could be cultured up to passage 8. Under our culture conditions, hiMGCs and pMGCs expressed low transcript levels of RLPB1, AQP4, KCNJ1, KCJN10, and SLC1A3. Using a disease modeling approach, we showed that hiMGCs could be used to model the features of diabetic retinopathy (DR)‐associated dyslipidemia. Indeed, palmitate, a major free fatty acid with elevated plasma levels in diabetic patients, induced the expression of inflammatory cytokines found in the ocular fluid of DR patients such as CXCL8 (IL‐8) and ANGPTL4. Moreover, the analysis of palmitate‐treated hiMGC secretome showed an upregulation of proangiogenic factors strongly related to DR, including ANG2, Endoglin, IL‐1β, CXCL8, MMP‐9, PDGF‐AA, and VEGF. Thus, hiMGCs could be an alternative to pMGCs and an extremely valuable tool to help to understand and model glial cell involvement in retinal disorders, including DR., Main Points We developed a protocol to generate and bank human iPSC‐derived Müller Glial cells (hiMGCs).hiMGCs showed phenotypic characteristics and transcriptomic profile of primary MGCs.hiMGCs can be used to model the features of diabetic retinopathy.

Glycogen Synthase Kinase 3 (GSK) proteins (GSK3α and GSK3β) are key mediators of signaling pathways, with crucial roles in coordinating fundamental biological processes during neural development. Here we show that the complete loss of GSK3 signaling in mouse retinal progenitors leads to microphthalmia with broad morphological defects. Both proliferation of retinal progenitors and neuronal differentiation are impaired and result in enhanced cell death. A single wild-type allele of either Gsk3α or Gsk3β is able to rescue these phenotypes. In this genetic context, all cell types are present with a functional retina. However, we unexpectedly detect a large number of cells in the inner nuclear layer expressing retinal ganglion cell (RGC)-specific markers (called displaced RGCs, dRGCs) when at least one allele of Gsk3α is expressed. Excess dRGCs lead to increased number of axons projecting into the ipsilateral medial terminal nucleus, an area of the brain belonging to the non-image-forming visual circuit and poorly targeted by RGCs in wild-type retina. Transcriptome analysis and optomotor response assay suggest that at least a subset of dRGCs in Gsk3 mutant mice are direction-selective RGCs. Our study thus uncovers a unique role of GSK3 in controlling the genesis of dRGCs, a rare and poorly characterized retinal cell type.

International audience; The effect of silicon on the natural sintering of Y3NbO7 powders is studied. Characterizations shows that Si increases densification rate and grain growth but it also leads to the apparition of a secondary silicate phase. The silicate precipitation results from the segregation of silicon favored by matter transport through a liquid phase. The distribution of the secondary phase have been studied by electronic microscopy and mapped by micro-luminescence. Thermodynamic calculations of Gibbs energy of the different phase as well as binary and ternary phase diagrams have been conducted to support interpretation of the sintering experimental results.

The retina has one of the highest energy demands in the human body, and proper regulation of metabolism among the cell types of the retina is required for functional vision. Recent studies have reported that retinal metabolism is disrupted during retinal degeneration and aging, while therapies designed to enhance metabolism can slow or prevent degeneration. Here, we show that multiple metabolic pathways, including those regulated by a key ATP sensor and regulator of metabolism, AMP-activated-kinase (AMPK), were dysregulated in a model of inherited retinal degeneration. In order to assess the direct role of AMPK in regulating retinal metabolism, we used mice with retina-specific knockout of AMPK activity. Conditional loss of AMPK resulted in impaired visual function at an early age, with slow photoreceptor loss observed in older mice. Moreover, we found that loss of AMPK resulted in decreased metabolic flux from glucose, decreased mitochondrial DNA copy number, decreased mitochondrial-related gene expression, and alterations in mitochondrial morphology in the photoreceptors, all of which preceded degeneration. Surprisingly, metabolic changes from the loss of AMPK in retinal neurons also resulted in secondary degeneration of retinal pigment epithelial (RPE) cells. Together, these data show that AMPK signaling is important for maintaining metabolic homeostasis of the retina and support the hypothesis that photoreceptors and RPE have a shared metabolic ecosystem that is controlled, in part, by AMPK.

An accurate description of brain white matter anatomy in vivo remains a challenge. However, technical progress allows us to analyze structural variations in an increasingly sophisticated way. Current methods of processing diffusion MRI data now make it possible to correct some limiting biases. In addition, the development of statistical learning algorithms offers the opportunity to analyze the data from a new perspective. We applied newly developed tractography models to extract quantitative white matter parameters in a group of patients with chronic temporal lobe epilepsy. Furthermore, we implemented a statistical learning workflow optimized for the MRI diffusion data - the TractLearn pipeline - to model inter-individual variability and predict structural changes in patients. Finally, we interpreted white matter abnormalities in the context of several other parameters reflecting clinical status, as well as neuronal and cognitive functioning for these patients. Overall, we show the relevance of such a diffusion data processing pipeline for the evaluation of clinical populations. The "global to fine scale" funnel statistical approach proposed in this study also contributes to the understanding of neuroplasticity mechanisms involved in refractory epilepsy, thus enriching previous findings.

Cone photoreceptor cell death in inherited retinal diseases, such as Retinitis Pigmentosa (RP), leads to the loss of high acuity and color vision and ultimately to blindness. In RP, a vast number of mutations perturb the structure and function of rod photoreceptors while cones remain initially unaffected. Cone death follows rod death secondarily due to increased oxidative stress, inflammation, and loss of structural and nutritional support provided by rods. Here, we show that secondary cone cell death in animal models for RP was associated with an increased activity of histone deacetylates (HDACs). A single intravitreal injection of an HDAC inhibitor at a late stage of the disease, when majority of rods have already degenerated, was sufficient to delay cone death and support long-term cone survival. Moreover, the surviving cones remained light sensitive and initiated light-driven ganglion cell responses. RNA-seq analysis of protected cones demonstrated that HDAC inhibition led to multi-level protectionviaregulation of different pro-survival pathways, including MAPK, PI3K-Akt, and autophagy. This study suggests a unique possibility for a targeted pharmacological protection of both primary degenerating rods and secondary dying cones by HDAC inhibition and creates hope to maintain vision in RP patients independent of the disease stage.

Glycogen synthase kinase 3 (GSK3) proteins (GSK3α and GSK3β) are key mediators of signaling pathways, with crucial roles in coordinating fundamental biological processes during neural development. Here we show that the complete loss of GSK3 signaling in mouse retinal progenitors leads to microphthalmia with broad morphologic defects. A single wild-type allele of eitherGsk3αorGsk3βis able to rescue this phenotype. In this genetic context, all cell types are present in a functional retina. However, we unexpectedly detected a large number of cells in the inner nuclear layer expressing retinal ganglion cell (RGC)-specific markers (called displaced RGCs, dRGCs) when at least one allele ofGsk3αis expressed. The excess of dRGCs leads to an increased number of axons projecting into the ipsilateral medial terminal nucleus, an area of the brain belonging to the non-image-forming visual circuit and poorly targeted by RGCs in wild-type retina. Transcriptome analysis and optomotor response assay suggest that at least a subset of dRGCs inGsk3mutant mice are direction-selective RGCs. Our study thus uncovers a unique role of GSK3 in controlling the production of ganglion cells in the inner nuclear layer, which correspond to dRGCs, a rare and poorly characterized retinal cell type.

Protein inhibitor of activated Stat 3 (Pias3) is implicated in guiding specification of rod and cone photoreceptors through post-translational modification of key retinal transcription factors. To investigate its role during retinal development, we deleted exon 2-5 of the mouse Pias3 gene, which resulted in complete loss of the Pias3 protein. Pias3−/− mice did not show any overt phenotype, and retinal lamination appeared normal even at 18 months. We detected reduced photopic b-wave amplitude by electroretinography following green light stimulation of postnatal day (P)21 Pias3−/− retina, suggesting a compromised visual response of medium wavelength (M) cones. No change was evident in response of short wavelength (S) cones or rod photoreceptors until 7 months. Increased S-opsin expression in the M-cone dominant dorsal retina suggested altered distribution of cone photoreceptors. Transcriptome profiling of P21 and 18-month-old Pias3−/− retina revealed aberrant expression of a subset of photoreceptor genes. Our studies demonstrate functional redundancy in SUMOylation-associated transcriptional control mechanisms and identify a specific, though limited, role of Pias3 in modulating spatial patterning and optimal function of cone photoreceptor subtypes in the mouse retina., Summary: Loss of Pias3 in mice results in altered dorso-ventral patterning of retinal cone photoreceptors by modulating the expression of a subset of genes, but does not affect rod development.

To compare the consequences of water hardness on canning of dry beans, baked beans were prepared using water that contained 70 or 260 ppm calcium for soaking/blanching of navy beans and in the preparation of a tomato sauce for brine. Another factor examined for this project involved increasing the initial temperature of the canned beans prior to thermal processing by eliminating the cooling step after blanching. In general, if the initial temperature prior to thermal processing can be increased, the process time (B

There is significant interest in mid-wavelength type II strained layer superlattices (SLSs) and HgCdTe material systems for background limited performance, operating at significantly higher temperature, T ≥ 150 K, than InSb, T ≈ 80–90 K. A precise knowledge of the electronic and optical properties of these materials is desirable since they determine detector performance and are needed for input parameters in self-consistent physics-based predictive models. Recently, data on the optical absorption coefficient, and the hole minority carrier lifetime has become available, suggesting that in the extrinsic region the limiting recombination processes in mid-wavelength type II Ga-free SLSs are radiative and Shockley–Read–Hall (SRH). These findings provide the opportunity for comparisons with mid-wavelength HgCdTe. The comparisons show that the radiative recombination coefficients are similar; however, the SRH lifetime limited to 9 μs for the SLS implies that the dark current density is expected to be limited by bulk generation–recombination (G–R) SRH processes for temperatures below 160 K; hence requiring heterojunction designs to suppress the G–R dark currents and be diffusion limited. Mid-wavelength infrared HgCdTe photodiodes are shallow p+n photovoltaic devices and because of the very long SRH hole lifetime are diffusion radiatively limited photodiodes down to 80 K.

Retinal dystrophies are a major cause of blindness for which there are currently no curative treatments. Transplantation of stem cell‐derived neuronal progenitors to replace lost cells has been widely investigated as a therapeutic option. Another promising strategy would be to trigger self‐repair mechanisms in patients, through the recruitment of endogenous cells with stemness properties. Accumulating evidence in the past 15 year0s has revealed that several retinal cell types possess neurogenic potential, thus opening new avenues for regenerative medicine. Among them, Müller glial cells have been shown to be able to undergo a reprogramming process to re‐acquire a stem/progenitor state, allowing them to proliferate and generate new neurons for repair following retinal damages. Although Müller cell–dependent spontaneous regeneration is remarkable in some species such as the fish, it is extremely limited and ineffective in mammals. Understanding the cellular events and molecular mechanisms underlying Müller cell activities in species endowed with regenerative capacities could provide knowledge to unlock the restricted potential of their mammalian counterparts. In this context, the present review provides an overview of Müller cell responses to injury across vertebrate model systems and summarizes recent advances in this rapidly evolving field. Developmental Dynamics 245:727–738, 2016. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc., Key findings The present review provides an overview of Müller cell responses to injury across vertebrate model systems and summarizes recent advances in this rapidly evolving field.

Summary: Contrasting with fish or amphibian, retinal regeneration from Müller glia is largely limited in mammals. In our quest toward the identification of molecular cues that may boost their stemness potential, we investigated the involvement of the Hippo pathway effector YAP (Yes-associated protein), which is upregulated in Müller cells following retinal injury. Conditional Yap deletion in mouse Müller cells prevents cell-cycle gene upregulation that normally accompanies reactive gliosis upon photoreceptor cell death. We further show that, in Xenopus, a species endowed with efficient regenerative capacity, YAP is required for their injury-dependent proliferative response. In the mouse retina, where Müller cells do not spontaneously proliferate, YAP overactivation is sufficient to induce their reprogramming into highly proliferative cells. Overall, we unravel a pivotal role for YAP in tuning Müller cell proliferative response to injury and highlight a YAP-EGFR (epidermal growth factor receptor) axis by which Müller cells exit their quiescence state, a critical step toward regeneration. : While fish and amphibian Müller cells behave as retinal stem cells upon injury, their regenerative potential is limited in mammals. Hamon et al. show that YAP is required for their cell-cycle re-entry in Xenopus and is sufficient in mouse to awake them from quiescence and trigger their proliferative response. Keywords: Müller cells, reactive gliosis, retinal regeneration, Hippo/YAP pathway, EGFR pathway

International audience; In the mammalian retina, rod and cone photoreceptors transmit the visual information to bipolar neurons through highly specialized ribbon synapses. We have limited understanding of regulatory pathways that guide morphogenesis and organization of photoreceptor presynaptic architecture in the developing retina. While neural retina leucine zipper (NRL) transcription factor determines rod cell fate and function, cone-rod homeobox (CRX) controls the expression of both rod- and cone-specific genes and is critical for terminal differentiation of photoreceptors. A comprehensive immunohistochemical evaluation of Crx-/- (null), CrxRip/+ and CrxRip/Rip (models of dominant congenital blindness) mouse retinas revealed abnormal photoreceptor synapses, with atypical ribbon shape, number and length. Integrated analysis of retinal transcriptomes of Crx-mutants with CRX- and NRL-ChIP-Seq data identified a subset of differentially expressed CRX target genes that encode presynaptic proteins associated with the cytomatrix active zone (CAZ) and synaptic vesicles. Immunohistochemistry of Crx-mutant retina validated aberrant expression of REEP6, PSD95, MPP4, UNC119, UNC13, RGS7 and RGS11, with some reduction in Ribeye and no significant change in immunostaining of RIMS1, RIMS2, Bassoon and Pikachurin. Our studies demonstrate that CRX controls the establishment of CAZ and anchoring of ribbons, but not the formation of ribbon itself, in photoreceptor presynaptic terminals.

Contrasting with fish or amphibian, retinal regeneration from Müller glial cells is largely limited in mammals. In our quest towards the identification of molecular cues that may boost their stemness potential, we investigated the involvement of the Hippo pathway effector YAP, which we previously found to be upregulated in Müller cells following retinal injury. We report that conditionalYapdeletion in Müller cells prevents the upregulation of cell cycle genes that normally accompanies reactive gliosis upon photoreceptor cell death. This occurs as a consequence of defective EGFR signaling. Consistent with a function of YAP in triggering Müller glia cell cycle re-entry, we further show that inXenopus, a species endowed with efficient regenerative capacity, YAP is required for their injury-dependent proliferative response. Finally, and noteworthy, we reveal that YAP overactivation in mouse Müller cells is sufficient to induce their reprogramming into highly proliferative cells. Overall, we unravel a pivotal role for YAP in tuning Müller cell response to injury and highlight a novel YAP-EGFR axis by which Müller cells exit their quiescence state, a critical step towards regeneration.

Chemokine signaling via CX3CL1-CX3CR1 helps regulate homeostasis in the population of microglia in the adult mouse retina., Microglia have been discovered to undergo repopulation following ablation. However, the functionality of repopulated microglia and the mechanisms regulating microglia repopulation are unknown. We examined microglial homeostasis in the adult mouse retina, a specialized neural compartment containing regular arrays of microglia in discrete synaptic laminae that can be directly visualized. Using in vivo imaging and cell-fate mapping techniques, we discovered that repopulation originated from residual microglia proliferating in the central inner retina that subsequently spread by centrifugal migration to fully recapitulate pre-existing microglial distributions and morphologies. Repopulating cells fully restored microglial functions including constitutive “surveying” process movements, behavioral and physiological responses to retinal injury, and maintenance of synaptic structure and function. Microglial repopulation was regulated by CX3CL1-CX3CR1 signaling, slowing in CX3CR1 deficiency and accelerating with exogenous CX3CL1 administration. Microglial homeostasis following perturbation can fully recover microglial organization and function under the regulation of chemokine signaling between neurons and microglia.

Retinal progenitor proliferation and differentiation are tightly controlled by extrinsic cues and distinctive combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we have characterized Bcl-2-associated transcription factor (Bclaf1) during rodent retinogenesis. Bclaf1 expression is restricted to early-born cell types, such as ganglion, amacrine, and horizontal cells. Analysis of developing retinas inBclaf1-deficient mice revealed a reduction in the numbers of retinal ganglion cells, amacrine cells and horizontal cells and an increase in the numbers of cone photoreceptor precursors. Silencing ofBclaf1expression byin vitroelectroporation of shRNA in embryonic retina confirmed that Bclaf1 serves to promote amacrine and horizontal cell differentiation. Misexpression of Bclaf1 in late retinal progenitors was not sufficient to directly induce the generation of amacrine and horizontal cells. Domain deletion analysis indicated that the N-terminal domain of Bclaf1 containing an arginine-serine-rich and a bZip domain is required for its effects on retinal cell differentiation. In addition, analysis revealed that Bclaf1 function occurs independently of its interaction with endogenous Bcl-2-related proteins. Altogether, our data demonstrates that Bclaf1expression in postmitotic early-born cells facilitates the differentiation of early retinal precursors into retinal ganglion cells, amacrine cells, and horizontal cells rather than into cone photoreceptors.

Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function.SIGNIFICANCE STATEMENTMicroglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.

Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor neural retina leucine zipper (NRL). The loss ofNrl(Nrl−/−) in mice results in a retina with predominantly S-opsin-containing cones that exhibit molecular and functional characteristics of wild-type cones. Here, we report thatNrl−/−retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by 4 months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic electroretinogram. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. TheNrl−/−mouse illustrates the long-term viability of cones in the absence of rods and retinal pigment epithelium defects in a rodless retina. We propose thatNrl−/−retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.

The orphan nuclear receptor NR2E3 is a direct transcriptional target of NRL, the key basic motif leucine zipper transcription factor that dictates rod versus cone photoreceptor cell fate in the mammalian retina. The lack of NR2E3 function in humans and in retinal degeneration rd7 mutant mouse leads to increased S-cones accompanied by rod degeneration, whereas ectopic expression of Nr2e3 in the cone-only Nrl(-/-) retina generates rod-like cells that do not exhibit any visual function. Using GFP to tag the newborn rods and by 5-bromo-2'-deoxyuridine birthdating, we demonstrate that early-born post-mitotic photoreceptor precursors in the rd7 retina express cone-specific genes. Transgenic mouse studies in the rd7 background show that Nr2e3 when expressed under the control of Crx promoter can restore rod photoreceptor function and suppress cone gene expression. Furthermore, Nr2e3 expression in photoreceptor precursors committed to be rods (driven by the Nrl promoter) could completely rescue the retinal phenotype of the rd7 mice. We conclude that excess of S-cones in the rd7 retina originate from photoreceptor precursors with a 'default' fate and not from proliferation of cones and that Nr2e3 is required to suppress the expression of S-cone genes during normal rod differentiation. These studies further support the 'transcriptional dominance' model of photoreceptor cell fate determination and provide insights into the pathogenesis of retinal disease phenotypes caused by NR2E3 mutations.

Ciliary neurotrophic factor (CNTF) participates in retinal development by inhibiting rod differentiation and promoting bipolar and Müller cell differentiation. In order to identify genes which are regulated by CNTF in the developing retina, we carried out a subtractive hybridization study. By this approach, we identified the Pleiotrophin (Ptn) as an upregulated gene in postnatal day 0 (P0) retinal explants upon addition of CNTF. Correlation of overall expression patterns between different retinal cell markers and Ptn in situ hybridization suggest that Ptn transcripts are initially expressed in progenitor cells then in postmitotic precursors of the INL expressing the Chx10 gene, and later in some differentiated retinal Müller glial (RMG) cells and rod–bipolar cells. Overexpression of Ptn by in vitro electroporation of P0 rat retinal explants partially blocks rod differentiation and promotes bipolar cell production, similar to effects of exogenous CNTF and leukemia inhibitory factor (LIF). Furthermore, in P0 retinal explants from mice lacking Ptn, the inhibitory effect of CNTF and LIF on rod differentiation is partially reduced and the cytokine-induced bipolar cell differentiation is largely prevented. Together, these results demonstrate that influence of CNTF family of cytokines on the differentiation of late retinal progenitor cell population is partially mediated by the release of Ptn.

We report here elements for functional characterization of two members of the Saccharomyces cerevisiae Ypt/Rab GTPase activating proteins family (GAP): Gyp5p, a potent GAP in vitro for Ypt1p and Sec4p, and the protein Ymr192wp/APP2 that we propose to rename Gyl1p (GYp like protein). Immunofluorescence experiments showed that Gyp5p and Gyl1p partly colocalize at the bud emergence site, at the bud tip and at the bud neck during cytokinesis. Subcellular fractionation and co-immunoprecipitation experiments showed that Gyp5p and Gyl1p co-fractionate with post-Golgi vesicles and plasma membrane, and belong to the same protein complexes in both localizations. We found by co-immunoprecipitation experiments that a fraction of Gyp5p interacts with Sec4p, a small GTPase involved in exocytosis, and that a fraction of Gyl1p associates at the plasma membrane with the Gyp5p/Sec4p complexes. We showed also that GYP5 genetically interacts with SEC2, which encodes the Sec4p exchange factor. Examination of the gyp5Δgyl1Δ mutants grown at 13°C revealed a slight growth defect, a secretion defect and an accumulation of secretory vesicles in the small-budded cells. These data suggest that Gyp5p and Gyl1p are involved in control of polarized exocytosis.

Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA, which is typically recessive; however, mutations in homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. The mechanism of CRX-associated LCA is not understood. Here, we identified a spontaneous mouse mutant with a frameshift mutation in Crx (CrxRip). We determined that CrxRip is a dominant mutation that results in congenital blindness with nonrecordable response by ERG and arrested photoreceptor differentiation with no associated degeneration. Expression of LCA-associated dominant CRX frameshift mutations in mouse retina mimicked the CrxRip phenotype, which was rescued by overexpression of WT CRX. Whole-transcriptome profiling using deep RNA sequencing revealed progressive and complete loss of rod differentiation factor NRL in CrxRip retinas. Expression of NRL partially restored rod development in CrxRip/+ mice. We show that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated in CrxRip mice and ectopic expression of OTX2 rescued the rod differentiation defect. Together, our data indicate that OTX2 maintains Nrl expression in developing rods to consolidate rod fate. Our studies provide insights into CRX mutation-associated congenital blindness and should assist in therapeutic design.

Antecedentes: El sangrado genital anormal es una causa frecuente de consulta en la adolescencia. En este período, la principal causa de sangrado es la metrorragia asociada a ciclos anovulatorios producto de la inmadurez del eje hipotálamo-hipofisario-gonadal. Dentro de las causas infrecuentes de sangrado genital anormal en ese período está el pólipo endometrial. Caso clínico: Presentamos el caso de una niña de 13 años cuya causa de sangrado correspondió a un pólipo endometrial, sospechado por ultrasonografía, resecado mediante histeroscopia y confirmado mediante estudio histológico. Discusión: Pese a su baja incidencia, los pólipos endometriales deben ser considerados como parte del diagnóstico diferencial en adolescentes que consultan por trastorno menstrual, particularmente en aquellos casos sin respuesta a la terapia hormonal y donde la ultrasonografía muestra engrosamiento endometrial. Background: Abnormal genital bleeding is a common cause of medical consultation in patients during adolescence. In this period, the main cause of genital bleeding is metrorrhagia in relation to anovulatory cycles due to immaturity of hypothalamus- pituitary-gonadal axis. Among the uncommon causes of bleeding at this age is the endometrial polyp. Case report: We report a 13 year old girl with abnormal uterine bleeding due to endometrial polyp, suspected during a pelvic ultrasound, removed by hysteroscopy, and confirmed by histological analysis. Discussion: Despite its low incidence, endometrial polyps should be included in the differential diagnosis of adolescents presenting menstrual disorders, particularly in those with no response to hormonal therapy and endometrial thickness in ultrasound.

The spectral functions for a linear chain show strong singularities when calculated with the usual perturbation-theory expressions. Results from molecular-dynamics simulations show no such signularities. We modify the perturbation-theory calculation to include lifetimes, and hence widths, for the intermediate phonons in the cubic term. The widths and shifts of the intermediate lines are assumed, for simplicity, to be proportional to their frequencies, and are calculated self-consistently. The agreement with the molecular-dynamics results is substantially improved.

International audience; Foxn4, a member of the N-family forkhead transcription factors, controls fate decision in mouse retina and spinal cord as well as in zebrafish heart. Analysis of Foxn4 amino acid sequence revealed the presence of a region homologous to the activation domain of its close relative Foxn1 in between C-terminal amino acids 402 and 455 of Foxn4 protein. The requirement of Foxn4 putative activation domain remains to be elucidated. Using a gain-of function approach in rat and chick retinal explants, we report that deletion of Foxn4 putative activation domain results in a complete loss of its activity during retinogenesis. Target promoter transcription assay indicates that this domain is critical for Foxn4 transcriptional regulatory properties in vitro. Accordingly, in chick retinal explants, this domain is required for proper regulation of target retinogenic factors expression by Foxn4. Thus, our study demonstrates that the domain between amino acids 402 and 455 is necessary for Foxn4 transcriptional activity both in vitro and in the retina.

Reversible image compression rarely achieves compression ratios larger than about 3:1. An explanation of this limit is offered, which hinges upon the additive noise the sensor introduces into the image. Simple models of this noise allow lower bounds on the bit rate to be estimated from sensor noise parameters rather than from ensembles of typical images. The model predicts that an 8-b single-band image subject to noise with unit standard deviation can be compressed reversibly to no less than 2.0 b/pixel, equivalent to a maximum compression ratio of about 4:1. The model has been extended to multispectral imagery. The Airborne Visible and Infra Red Imaging Spectrometer (AVIRIS) is used as an example, as the noise in its 224 bands is well characterized. The model predicts a lower bound on the bit rate for the compressed data of about 5.5 b/pixel when a single codebook is used to encode all the bands. A separate codebook for each band (i.e., 224 codebooks) reduces this bound by 0.5 b/pixel to about 5.0 b/pixel, but 90% of this reduction is provided by only four codebooks. Empirical results corroborate these theoretical predictions. >

Se presenta el caso de una paciente de 56 años que ingresó al hospital por una historia caracterizada por decaimiento, constipación, fractura reciente de radio, hipercalcemia de 18 mg/dl, hipofosfemia de 1,2 meq/ dl y elevación de paratohormona de 995 pg/ml. Además, presentaba un nódulo tiroídeo de 6 cm. Posterior al tratamiento médico de la hipercalcemia, se realizó el tratamiento quirúrgico con el diagnóstico definitivo de hiperplasia paratiroídea multiglandular con una de ellas en ubicación intratiroídea. We report a 56 years old female, admitted to the hospital for the study of general weakness, constipation, a recent radial fracture, hypercalcemia of 18 mg/dl, hypophosphatemia of 1.2 meq/dl and a serum parathormone (PTH) of 995 pg/ml. A thyroid ultrasound disclosed a nodule of 6 cm diameter. Following emergency management and normalization of the hypercalcemia, that patient was subjected to a total thyroidectomy and parathyroidectomy. The pathological study of the surgical piece showed a multiglandular hyperplasia of the parathyroid glands. One of these was located inside the left thyroid lobe.