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Purpose:Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML), where ITD mutations in tyrosine kinase receptor FLT3 are associated with poor prognostic outcomes. Several FLT3 inhibitors (FLT3i) are in clinical trials for high-risk FLT3-ITD–positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence of FLT3-ITD mutations might not guarantee effective clinical response. Motivated by the heterogeneity of FLT3-ITD mutations, we investigated the effects of FLT3-ITD structural features on the response of AML patients to treatment.Experimental Design: We developed the HeatITup (HEAT diffusion for Internal Tandem dUPlication) algorithm to identify and quantitate ITD structural features including nucleotide composition. Using HeatITup, we studied the impact of ITD structural features on the clinical response to FLT3i and induction chemotherapy in FLT3-ITD–positive AML patients.Results:HeatITup accurately identifies and classifies ITDs into newly defined categories of “typical” or “atypical” based on their nucleotide composition. A typical ITD's insert sequence completely matches the wild-type FLT3, whereas an atypical ITD's insert contains nucleotides exogenous to the wild-type FLT3. Our analysis shows marked divergence between typical and atypical ITD mutation features. Furthermore, our data suggest that AML patients carrying typical FLT3-ITDs benefited significantly more from both FLT3i and induction chemotherapy treatments than patients with atypical FLT3-ITDs.Conclusions:These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing toward personalized treatment of AML patients, and enable researchers and clinicians to unravel ITD complexity using the provided software.See related commentary by Gallipoli and Huntly, p. 460

Figure S1: Characterization of FLT3-ITD clones in the FLT3i cohort. Table S1: Confusion matrix of HeatITup and genetic reviewers manual annotation for sequences in Figure 3. Table S2: Univariate Cox regression analyses for the FLT3-ITD-positive UPENN cohort. Table S3: Cox regression analysis of FLT3-ITD duplication length for the non-FLT3i-treated cohort. Table S4: Cox regression analysis of FLT3-ITD classification for the non-FLT3i-treated cohort. Table S5: Cox regression analysis of FLT3-ITD duplication length with classification for the non-FLT3i-treated cohort. Table S6: Cox regression analysis of FLT3-ITD classification for the top third longest ITD duplications in the non-FLT3i-treated cohort (Benjamini-Hochberg adjusted p-values from long non-FLT3i-treated cohort analyses). Table S7: Cox regression analysis of FLT3-ITD spacer sequence exogenous fraction for the non-FLT3i-treated cohort. Table S8: Cox regression analysis of FLT3-ITD spacer sequence exogenous fraction for the top third longest ITD duplications in the non-FLT3i-treated cohort. Table S9: Cox regression analysis of FLT3i treatment for the UPENN cohort. Table S10: Cox regression analysis of FLT3-ITD duplication length for the FLT3i-treated cohort (Benjamini-Hochberg adjusted p-values from FLT3i cohort analyses). Table S11: Cox regression analysis of FLT3-ITD classification for the FLT3i-treated cohort (Benjamini-Hochberg adjusted p-values from FLT3i cohort analyses). Table S12: Cox regression analysis of FLT3-ITD spacer sequence exogenous fraction for the FLT3i-treated cohort.

Retinal pigment epithelium (RPE) at the back of the eye is a monolayer of cells with an extensive network of gap junctions that contributes to retinal health in a multitude of ways. One of those roles is the phagocytosis of photoreceptor outer segments. This renewal is under circadian regulation and peaks after light onset. Connexin 43 (Cx43) is the most predominantly expressed gap junction protein in RPE. In this study, we examine how gap junctions and specifically, Cx43 phosphorylation, contribute to phagocytosis in both human embryonic stem cell derived RPE and mouse RPE monolayers. We show that both Rac1 and CDK5 have differences in protein localization at different points in phagocytosis, and that by using their effectors, the capability of RPE for phagocytosis changes. CDK5 has not yet been reported in RPE tissue, and here we show that it likely regulates Cx43 localization and resulting electrical coupling. We find that gap junctions in RPE are temporally highly dynamic during phagocytosis and that regulation of gap junctions via phosphorylation is likely critical for maintaining eye health.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs, we developed AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX show the superior performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulated known islet pathobiology and showed differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+T cells infiltration in islets during type 1 diabetes progression.

Primates explore their visual environment by making frequent saccades, discrete and ballistic eye movements that direct the fovea to specific regions of interest. Saccades produce large and rapid changes in input. The magnitude of these changes and the limited signaling range of visual neurons mean that effective encoding requires rapid adaptation. Here, we explore how macaque cone photoreceptors maintain sensitivity under these conditions. Adaptation makes cone responses to naturalistic stimuli highly nonlinear and dependent on stimulus history. Such responses cannot be explained by linear or linear-nonlinear models but are well explained by a biophysical model of phototransduction based on well-established biochemical interactions. The resulting model can predict cone responses to a broad range of stimuli and enables the design of stimuli that elicit specific (e.g., linear) cone photocurrents. These advances will provide a foundation for investigating the contributions of cone phototransduction and post-transduction processing to visual function.SIGNIFICANCE STATEMENTWe know a great deal about adaptational mechanisms that adjust sensitivity to slow changes in visual inputs such as the rising or setting sun. We know much less about the rapid adaptational mechanisms that are essential for maintaining sensitivity as gaze shifts around a single visual scene. We characterize how phototransduction in cone photoreceptors adapts to rapid changes in input similar to those encountered during natural vision. We incorporate these measurements into a quantitative model that can predict cone responses across a broad range of stimuli. This model not only shows how cone phototransduction aids the encoding of natural inputs but also provides a tool to identify the role of the cone responses in shaping those of downstream visual neurons.

Obtaining a parts list of the sensory components of the retina is vital to understanding the effects of light in behavior, health, and disease. Rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) are the best described photoreceptors in the mammalian retina, but recent functional roles have been proposed for retinal neuropsin (Opn5) - an atypical opsin. However, little is known about the pattern of Opn5 expression in the retina. Using cre (Opn5cre ) and cre-dependent reporters, we uncover patterns of Opn5 expression and find that Opn5 is restricted to retinal ganglion cells (RGCs). Opn5-RGCs are non-homogenously distributed through the retina, with greater densities of cells located in the dorsotemporal quadrant. In addition to local topology of these cells, using cre-dependent AAV viral tracing, we surveyed their central targets and found that they are biased towards image-forming and image-stabilizing regions. Finally, molecular and electrophysiological profiling reveal that Opn5-RGCs comprise previously defined RGC types which respond optimally to edges and object-motion (F-mini-ONs, HD2, HD1, LEDs, ooDSRGCs, etc.). Together, these data describe the second collection of RGCs that express atypical opsins in the mouse, and expand the roles of image-forming cells in retinal physiology and function. This article is protected by copyright. All rights reserved.

A stimulus can be encoded in a population of spiking neurons through any change in the statistics of the joint spike pattern, yet we commonly summarize single-trial population activity by the summed spike rate across cells: the population peri-stimulus time histogram (pPSTH). For neurons with low baseline spike rate that encode a stimulus with a rate increase, this simplified representation works well, but for populations with high baseline rates and heterogeneous response patterns, the pPSTH has limited utility in capturing the neural representation of the stimulus. We simulated populations of spiking neurons that varied in size, baseline rate, burst statistics, and correlation, and we measured how these populations represent decreases (gaps) in spike rate. We introduce a different representation of the population spike pattern which we call an “information train,” and we show that it is more flexible and robust than the pPSTH in capturing stimulus information across different types of neuronal populations. In particular, we use this tool to study populations with varying levels of burstiness in their spiking statistics. We find that there is an optimal level of burstiness for gap detection that is robust to several other parameters of the population. Next, we consider this theoretical result in the context of experimental data from different types of retinal ganglion cells and determine that the baseline spike statistics of a particular, recently identified type support nearly optimal detection of both the onset and strength of a contrast step.

SummaryReceptive fields (RFs) are a foundational concept in sensory neuroscience. The RF of a sensory neuron is shaped by the properties of its synaptic inputs from connected neurons. In the early visual system, retinotopic maps define a strict relationship between the location of a cell’s dendrites and its RF location in visual space1–3. Retinal ganglion cells (RGCs), the output cells of the retina, form dendritic mosaics that tile retinal space and have corresponding RF mosaics that tile visual space1,2. The precise location of dendrites in some RGCs has been shown to predict their RF shape4. Previously described ON-OFF RGCs have aligned dendrites in ON and OFF synaptic layers, so the cells respond to increments and decrements of light at the same locations in visual space5–8. Here we report a systematic offset between the ON and OFF RFs of an RGC type. Surprisingly, this property does not come from offset ON and OFF dendrites but instead arises from electrical synapses with RGCs of a different type. This circuit represents a new channel for direct communication between ON and OFF RGCs. Using a multi-cell model, we find that offset ON-OFF RFs could improve the precision with which edge location is represented in an RGC population.

The output of spiking neurons depends both on their synaptic inputs and on their intrinsic properties. Retinal ganglion cells (RGCs), the spiking projection neurons of the retina, comprise over 40 different types in mice and other mammals, each tuned to different features of visual scenes. The circuits providing synaptic input to different RGC types to drive feature selectivity have been studied extensively, but there has been substantially less research aimed at understanding how the intrinsic properties of RGCs differ and how those differences impact feature selectivity. Here, we introduce an RGC type in the mouse, the Bursty Suppressed-by-Contrast (bSbC) RGC, whose contrast selectivity is shaped by its intrinsic properties. Surprisingly, when we compare the bSbC RGC to the OFF sustained alpha (OFFsA) RGC that receives similar synaptic input, we find that the two RGC types exhibit starkly different responses to an identical stimulus. We identified spike generation as the key intrinsic property behind this functional difference; the bSbC RGC undergoes depolarization block in conditions where the OFFsA RGC maintains a high spike rate. Pharmacological experiments, imaging, and compartment modeling demonstrate that these differences in spike generation are the result of differences in voltage-gated sodium channel conductances. Our results demonstrate that differences in intrinsic properties allow these two RGC types to detect and relay distinct features of an identical visual stimulus to the brain.

Summary Color vision, originating with opponent processing of spectrally distinct photoreceptor signals, plays important roles in animal behavior.1, 2, 3, 4 Surprisingly, however, comparatively little is understood about color processing in the brain, including in widely used laboratory mammals such as mice. The retinal gradient in S- and M-cone opsin (co-)expression has traditionally been considered an impediment to mouse color vision.5, 6, 7, 8 However, recent data indicate that mice exhibit robust chromatic discrimination within the central-upper visual field.9 Retinal color opponency has been reported to emerge from superimposing inhibitory surround receptive fields on the cone opsin expression gradient, and by introducing opponent rod signals in retinal regions with sparse M-cone opsin expression.10, 11, 12, 13 The relative importance of these proposed mechanisms in determining the properties of neurons at higher visual processing stages remains unknown. We address these questions using multielectrode recordings from the lateral geniculate nucleus (LGN) in mice with altered M-cone spectral sensitivity (Opn1mwR) and multispectral stimuli that allow selective modulation of signaling by individual opsin classes. Remarkably, we find many (∼25%) LGN cells are color opponent, that such cells are localized to a distinct medial LGN zone and that their properties cannot simply be explained by the proposed retinal opponent mechanisms. Opponent responses in LGN can be driven solely by cones, independent of cone-opsin expression gradients and rod input, with many cells exhibiting spatially congruent antagonistic receptive fields. Our data therefore suggest previously unidentified mechanisms may support extensive and sophisticated color processing in the mouse LGN., Highlights • Color opponency is common among neurons within a chromatic zone in the mouse LGN • Cone inputs to LGN drive color opponency across mesopic to photopic light levels • Cone inputs to opponent neurons derive from central and upper visual field • Subsets of LGN neurons display opponency for small or large stimuli only, Mouland et al. examine color processing in the mouse visual thalamus. They show that color opponency is widespread among cells within a specific zone of the lateral geniculate nucleus and that cones drive such responses, independent of cone-opsin expression gradients and rod input, providing a robust capacity for color discrimination in mice.

Retinal ganglion cells can be classified into more than 40 distinct subtypes, whether by functional classification or transcriptomics. The examination of these subtypes in relation to their physiology, projection patterns, and circuitry would be greatly facilitated through the identification of specific molecular identifiers for the generation of transgenic mice. Advances in single cell transcriptomic profiling have enabled the identification of molecular signatures for cellular subtypes that are only rarely found. Therefore, we used single cell profiling combined with hierarchical clustering and correlate analyses to identify genes expressed in distinct populations of Parvalbumin-expressing cells and functionally classified RGCs. RGCs were manually isolated based either upon fluorescence or physiological distinction through cell-attached recordings. Microarray hybridization and RNA-Sequencing were employed for the characterization of transcriptomes and in situ hybridization was utilized to further characterize gene candidate expression. Gene candidates were identified based upon cluster correlation, as well as expression specificity within physiologically distinct classes of RGCs. Further, we identified Prph, Ctxn3, and Prkcq as potential candidates for ipRGC classification in the murine retina. The use of these genes, or one of the other newly identified subset markers, for the generation of a transgenic mouse would enable future studies of RGC-subtype specific function, wiring, and projection.

Purpose: Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML), where ITD mutations in tyrosine kinase receptor FLT3 are associated with poor prognostic outcomes. Several FLT3 inhibitors (FLT3i) are in clinical trials for high-risk FLT3-ITD–positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence of FLT3-ITD mutations might not guarantee effective clinical response. Motivated by the heterogeneity of FLT3-ITD mutations, we investigated the effects of FLT3-ITD structural features on the response of AML patients to treatment. Experimental Design: We developed the HeatITup (HEAT diffusion for Internal Tandem dUPlication) algorithm to identify and quantitate ITD structural features including nucleotide composition. Using HeatITup, we studied the impact of ITD structural features on the clinical response to FLT3i and induction chemotherapy in FLT3-ITD–positive AML patients. Results: HeatITup accurately identifies and classifies ITDs into newly defined categories of “typical” or “atypical” based on their nucleotide composition. A typical ITD's insert sequence completely matches the wild-type FLT3, whereas an atypical ITD's insert contains nucleotides exogenous to the wild-type FLT3. Our analysis shows marked divergence between typical and atypical ITD mutation features. Furthermore, our data suggest that AML patients carrying typical FLT3-ITDs benefited significantly more from both FLT3i and induction chemotherapy treatments than patients with atypical FLT3-ITDs. Conclusions: These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing toward personalized treatment of AML patients, and enable researchers and clinicians to unravel ITD complexity using the provided software. See related commentary by Gallipoli and Huntly, p. 460

Purpose To assess retinal microvascular reactivity during dark adaptation and the transition to ambient light and after flicker stimulation using optical coherence tomography angiography (OCTA). Methods Fifteen eyes of 15 healthy participants were dark adapted for 45 minutes followed by OCTA imaging in the dark-adapted state. After 5 minutes of normal lighting, subjects underwent OCTA imaging. Participants were then subjected to a flashing light-emitting diode (LED) light and repeat OCTA. Parafoveal vessel density and adjusted flow index (AFI) were calculated for superficial (SCP), middle (MCP), and deep capillary plexuses (DCP), and then compared between conditions after adjusting for age, refractive error, and scan quality. SCP vessel length density (VLD) was also evaluated. Between-condition capillary images were aligned and subtracted to identify differences. We then analyzed images from 10 healthy subjects during the transition from dark adaptation to ambient light. Results SCP vessel density was significantly higher while SCP VLD was significantly lower during ambient light and flicker compared to dark adaptation. There was a significant positive mean value for DCP "flicker minus dark or light," suggesting more visible vessels during flicker due to changes in flow, dilation, or vessel recruitment. We found a significant, transient increase in SCP and decrease in both MCP and DCP vessel density during the transition from dark to light. Conclusions We show evidence suggesting constriction of deeper vessels and dilation of large SCP vessels during the transition from dark to light. This contrasts to redistribution of blood flow to deeper layers during dark adaptation and flicker stimulation.

Classification and characterization of neuronal types are critical for understanding their function and dysfunction. Neuronal classification schemes typically rely on measurements of electrophysiological, morphological, and molecular features, but aligning such datasets has been challenging. Here, we present a unified classification of mouse retinal ganglion cells (RGCs), the sole retinal output neurons. We used visually-evoked responses to classify 1859 mouse RGCs into 42 types. We also obtained morphological or transcriptomic data from subsets and used these measurements to align the functional classification to publicly available morphological and transcriptomic data sets. We created an online database that allows users to browse or download the data and to classify RGCs from their light responses using a machine learning algorithm. This work provides a resource for studies of RGCs, their upstream circuits in the retina, and their projections in the brain, and establishes a framework for future efforts in neuronal classification and open data distribution.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells destroy insulin-producing beta cells. The aetiology of this complex disease is dependent on the interplay of multiple heterogeneous cell types in the pancreatic environment. Here, we provide a single-cell atlas of pancreatic islets of 24 T1D, autoantibody-positive and nondiabetic organ donors across multiple quantitative modalities including ~80,000 cells using single-cell transcriptomics, ~7,000,000 cells using cytometry by time of flight and ~1,000,000 cells using in situ imaging mass cytometry. We develop an advanced integrative analytical strategy to assess pancreatic islets and identify canonical cell types. We show that a subset of exocrine ductal cells acquires a signature of tolerogenic dendritic cells in an apparent attempt at immune suppression in T1D donors. Our multimodal analyses delineate cell types and processes that may contribute to T1D immunopathogenesis and provide an integrative procedure for exploration and discovery of human pancreatic function.

Gap junctions are intercellular channels that permit the transfer of ions and small molecules between adjacent cells. These cellular junctions are particularly dense in the retinal pigment epithelium (RPE), and their contribution to many retinal diseases has been recognized. While gap junctions have been implicated in several aspects of RPE physiology, their role in shaping the electrical properties of these cells has not been characterized in mammals. The role of gap junctions in the electrical properties of the RPE is particularly important considering the growing appreciation of RPE as excitable cells containing various voltage-gated channels. We used a whole-cell patch clamp to measure the electrical characteristics and connectivity between RPE cells, both in cultures derived from human embryonic stem cells and in the intact RPE monolayers from mouse eyes. We found that the pharmacological blockade of gap junctions eliminated electrical coupling between RPE cells, and that the blockade of gap junctions or Cx43 hemichannels significantly increased their input resistance. These results demonstrate that gap junctions function in the RPE not only as a means of molecular transport but also as a regulator of electrical excitability. publishedVersion

Primates explore their visual environment by making frequent saccades, discrete and ballistic eye movements that direct the fovea to specific regions of interest. Saccades produce large and rapid changes in input. The magnitude of these changes and the limited signaling range of visual neurons means that effective encoding requires rapid adaptation. Here, we explore how cone photoreceptors maintain sensitivity under these conditions. Adaptation makes cone responses to naturalistic stimuli highly nonlinear and dependent on stimulus history. Such responses cannot be explained by linear or linear-nonlinear models but are well explained by a biophysical model of phototransduction with fast and slow adaptational mechanisms. The resulting model can predict cone responses to a broad range of stimuli and enables the design of stimuli that elicit specific (e.g. linear) cone photocurrents. These advances will provide a foundation for investigating the contributions of cones and post-cone processing to visual function.

Type 1 diabetes (T1D) is an autoimmune disease of only partially defined etiology in which immune cells destroy insulin-producing beta cells. Using single-cell transcriptomics and an advanced analytical strategy to assess pancreatic islets of T1D, autoantibody-positive, and non-diabetic organ donors, we identified both canonical cell types and rare insulin-expressing cells with a hybrid mixture of endocrine and exocrine gene signatures within all donors. We further found elevated expression of MHC Class II pathway genes in exocrine ductal cells of T1D donors, which we confirmed through CyTOF, in situ imaging mass cytometry, and immunofluorescence analysis. Taken together, our multimodal analyses identify novel cell types and processes that may contribute to T1D immunopathogenesis and provide new cellular and molecular insights into human pancreas function.

Tumors depend on a blood supply to deliver oxygen and nutrients, making tumor vasculature an attractive anticancer target. However, only a fraction of patients with cancer benefit from angiogenesis inhibitors. Whether antiangiogenic therapy would be more effective if targeted to individuals with specific tumor characteristics is unknown. To better characterize the tumor vascular environment both within and between cancer types, we developed a standardized metric - the endothelial index (EI) - to estimate vascular density in over 10,000 human tumors, corresponding to 31 solid tumor types, from transcriptome data. We then used this index to compare hyper- and hypovascular tumors, enabling the classification of human tumors into 6 vascular microenvironment signatures (VMSs) based on the expression of a panel of 24 vascular "hub" genes. The EI and VMS correlated with known tumor vascular features and were independently associated with prognosis in certain cancer types. Retrospective testing of clinical trial data identified VMS2 classification as a powerful biomarker for response to bevacizumab. Thus, we believe our studies provide an unbiased picture of human tumor vasculature that may enable more precise deployment of antiangiogenesis therapy.

Diabetic retinopathy (DR) has traditionally been viewed as either a microvasculopathy or a neuropathy, though neurovascular coupling deficits have also been reported and could potentially be the earliest derangement in DR. To better understand neurovascular coupling in the diabetic retina, we investigated retinal hemodynamics by optical coherence tomography angiography (OCTA) in individuals with diabetes mellitus (DM) but without DR (DM no DR) and mild non-proliferative DR (mild NPDR) compared to healthy eyes. Using an experimental design to monitor the capillary responses during transition from dark adaptation to light, we examined 19 healthy, 14 DM no DR and 11 mild NPDR individuals. We found that the only structural vascular abnormality in the DM no DR group was increased superficial capillary plexus (SCP) vessel density (VD) compared to healthy eyes, while mild NPDR eyes showed significant vessel loss in the SCP at baseline. There was no significant difference in inner retinal thickness between the groups. During dark adaptation, the deep capillary plexus (DCP) VD was lower in mild NPDR individuals compared to the other two groups, which may leave the photoreceptors more susceptible to ischemia in the dark. When transitioning from dark to ambient light, both diabetic groups showed a qualitative reversal of VD trends in the SCP and middle capillary plexus (MCP), with significantly decreased SCP at 5 min and increased MCP VD at 50 s compared to healthy eyes, which may impede metabolic supply to the inner retina during light adaptation. Mild NPDR eyes also demonstrated DCP dilation at 50 s and 5 min and decreased adjusted flow index at 5 min in light. Our results show altered neurovascular responses in all three macular vascular plexuses in diabetic subjects in the absence of structural neuronal changes on high resolution imaging, suggesting that neurovascular uncoupling may be a key mechanism in the early pathogenesis of DR, well before the clinical appearance of vascular or neuronal loss.

In the mouse, retinal output is computed by over 40 distinct types of retinal ganglion cells (RGCs) (Baden et al., 2016). Determining which of these many RGC types project to a retinorecipient region is a key step in elucidating the role that region plays in visually-mediated behaviors. Combining retrograde viral tracing and single-cell electrophysiology, we identify the RGC types which project to the olivary pretectal nucleus (OPN), a major visual structure. We find that retinal input to the OPN consists of a variety of intrinsically-photosensitive and conventional RGC types, the latter a diverse set of mostly ON RGCs. Surprisingly, while the OPN is most associated with the pupillary light reflex (PLR) pathway, requiring information about absolute luminance, we show that the majority of the retinal input to the OPN is from single cell type which transmits information unrelated to luminance. This ON-transient RGC accounts for two-thirds of the input to the OPN, and responds to small objects across a wide range of speeds. This finding suggests a role for the OPN in visually-mediated behaviors beyond the PLR.Significance statementThe olivary pretectal nucleus is a midbrain structure which receives direct input from retinal ganglion cells (RGC), and modulates pupil diameter in response to changing absolute light level. In the present study, we combine viral tracing and electrophysiology to identify the RGC types which project to the OPN. Surprisingly, the majority of its input comes from a single type which does not encode absolute luminance, but instead responds to small objects across a wide range of speeds. These findings are consistent with a role for the OPN apart from pupil control and suggest future experiments to elucidate its full role in visually-mediated behavior.

Purpose To test the hypothesis that hyperglycemia perturbs neurovascular\ coupling and compromises retinal vascular response during transition from dark to light in healthy subjects using optical coherence tomography angiography (OCTA). Methods Ten eyes of 10 healthy subjects were tested, first during fasting and then after receiving a 75-g oral glucose solution. In both sessions, OCTA imaging was done in the dark-adapted state and at 50 seconds, 2 minutes, 5 minutes, and 15 minutes of ambient light. Parafoveal vessel density (VD) and adjusted flow index (AFI) were calculated for the superficial capillary plexus (SCP), middle capillary plexus (MCP), and deep capillary plexus (DCP), and vessel length density was calculated for the SCP. These measurements were compared among conditions after adjusting for age, refractive error, and OCTA scan quality. Results Hyperglycemia leads to a complete reversal of dark/light adaptation trends in VD and AFI in all layers of the inner retina. In the dark, there is significantly decreased VD in the DCP in hyperglycemia. With a transition to light in hyperglycemia, we observed decreased VD in the SCP, increased vessel density in the MCP and DCP, and decreased AFI in all three layers. Conclusions Our results show that hyperglycemia significantly disrupts neurovascular coupling in healthy eyes, with potential metabolic deficits affecting photoreceptor oxygen demands during dark adaptation and the inner retina during light exposure. In pathological states, such as diabetic retinopathy, where the vasculature is already attenuated, retinal neurons may be exquisitely vulnerable to intermittent hyperglycemic challenge, which should be the focus of future studies.

Rome et al. show that Trib1 controls effector versus exhausted T cell differentiation and function by restraining effector programming during persistent infection, revealing a new regulatory axis that could be targeted to recover waning T cell responses during chronic disease., In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity., Graphical Abstract

Retinal ganglion cells (RGCs) were one of the first classes of sensory neurons to be described in terms of a receptive field (RF). Over the last six decades, our understanding of the diversity of RGC types and the nuances of their response properties has grown exponentially. We will review the current understanding of RGC receptive fields (RFs) mostly from studies in mammals, but including work from other vertebrates as well. We will argue for a new paradigm that embraces the fluidity of RGC RFs with an eye toward the neuroethology of vision. Specifically, we will focus on (1) different methods for measuring RGC RFs, (2) RF models, (3) feature selectivity and the distinction between fluid and stable RF properties, and (4) ideas about the future of understanding RGC RFs.

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks-one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.

Serine is the only amino acid that is encoded by two disjoint codon sets (TCN & AGY) so that a tandem substitution of two nucleotides is required to switch between the two sets. We show that these codon sets underlie distinct substitution patterns at positions subject to purifying and diversifying selections. We found that in humans, positions that are conserved among ~100 vertebrates, and thus subjected to purifying selection, are enriched for substitutions involving serine (TCN, denoted S′), proline, and alanine, (S′PA). In contrast, the less conserved positions are enriched for serine encoded with AGY codons (denoted S″), glycine and asparagine, (GS″N). We tested this phenomenon in the HIV envelope glycoprotein (gp120), and the V-gene that encodes B-cell receptors/antibodies. These fast evolving proteins both have hypervariable positions, which are under diversifying selection, closely adjacent to highly conserved structural regions. In both instances, we identified an opposite abundance of two groups of serine substitutions, with enrichment of S′PA in the conserved positions, and GS″N in the hypervariable regions. Finally, we analyzed the substitutions across 60,000 individual human exomes to show that, when serine has a specific functional constraint of phosphorylation capability, S′ codons are 32-folds less prone than S″ to substitutions to Threonine or Tyrosine that could potentially retain the phosphorylation site capacity. Combined, our results, that cover evolutionary signals at different temporal scales, demonstrate that through its encoding by two codon sets, serine allows for the existence of alternating substitution patterns within positions of functional maintenance versus sites of rapid diversification.

Transcriptional programs contribute to phenotypic and functional cell states. While elucidation of cell state heterogeneity and its role in biology and pathobiology has been advanced by studying single cell level measurements, the underlying assumptions of current analytical methods limit the identification and exploration of cell clades. Unlike other methods, which produce a single uni-layer partition of cells ignoring echelons of cell states, we presentTooManyCells, a software consisting of a suite of graph-based tools for efficient, global, and unbiased identification and visualization of cell clades while maintaining and presenting the relationship between cell states.TooManyCellsprovides a set of tools based on a matrix-free efficient divisive hierarchical spectral clustering algorithm wholly different from the prevalent Louvain-based methods.BirchBeer, the visualization component ofTooManyCells, introduces a new approach for single cell analysis that is built on a concept intentionally orthogonal to the widely used dimensionality reduction methods. Together, this suite of tools provide a paradigm shift in the analysis and interpretation of single cell data by enabling simultaneous comparisons of cell states at context-and application-dependent scales. A byproduct of this shift is the immediate detection and visualization of rare populations that outperforms previous algorithms as demonstrated by applying these tools to existing single cell RNA-seq data sets from various mouse organs.

SUMMARY Emerging single-cell epigenomic assays are used to investigate the heterogeneity of chromatin activity and its function. However, identifying cells with distinct regulatory elements and clearly visualizing their relationships remains challenging. To this end, we introduce TooManyPeaks to address the need for the simultaneous study of chromatin state heterogeneity in both rare and abundant subpopulations. Our analyses of existing data from three widely used single-cell assays for transposase-accessible chromatin using sequencing (scATAC-seq) show the superior performance of TooManyPeaks in delineating and visualizing pure clusters of rare and abundant subpopulations. Furthermore, the application of TooManyPeaks to new scATAC-seq data from drug-naive and drug-resistant leukemic T cells clearly visualizes relationships among these cells and stratifies a rare “resistant-like” drug-naive sub-clone with distinct cis-regulatory elements., Graphical Abstract, In brief Schwartz et al. present TooManyPeaks, a suite of algorithms to explore and visualize heterogeneity of regulatory elements by using single-cell ATAC-seq data. Using TooManyPeaks’s functionalities, they find evidence that heterogeneity of the chromatin accessibility state contributes to the propensity of Notch-mutated T leukemic cells to develop resistance to Notch inhibitors.

We present a new method for finding relationships of metadata labels using other data.We present a novel measure of aggregation within a hierarchical container.Our clumpiness measure is stable across tree size, label size, and numbers of labels.We quantify relationships of immune receptor V genes from their sequence fragments. When finding relationships in biological systems, we often describe hierarchies based on one facet of the data. However, when using this hierarchy to elucidate relationships between metadata, the distribution of metadata labels within the hierarchy may exhibit different levels of aggregation-uniform, random, or clumped. As of now, there exists no measure for finding the level of aggregation, or "clumpiness", between labels distributed among the leaves of a hierarchical container. We propose a clumpiness measure to aid in the quantification of relationships between metadata. We validated our measure with random trees and found that the measure is resistant to changes in the tree size, label size, and the number of types of labels, compared to the closest alternative measures. We used our clumpiness measure to quantify the relationships between light and heavy chains in human and mouse B cell and T cell receptor V genes based on their motifs. We found that the B cell heavy chains were the most aggregated while the T cell chains were the least aggregated and that the IGL chain was clumped the most with the T cell chains out of all of the B cell chains.

Purpose The purpose of this study was to investigate the acute effects of caffeine on retinal hemodynamics during dark to light adaptation using optical coherence tomography angiography (OCTA). Methods Thirteen healthy individuals (13 eyes) underwent OCTA imaging after dark adaptation and at repeated intervals during the transition to ambient light in two imaging sessions: control and after ingesting 200 mg of caffeine. We analyzed the parafoveal vessel density (VD) and adjusted flow index (AFI) of the superficial capillary plexus (SCP), middle capillary plexus (MCP), and deep capillary plexus (DCP), as well as the vessel length density (VLD) of the SCP. After adjusting for age, refractive error, and scan quality, we compared parameters between control and caffeine conditions. Results In the dark, MCP VD decreased significantly after caffeine (-2.63 ± 1.28%). During the transition to light, initially, DCP VD increased (12.55 ± 2.52%), whereas SCP VD decreased (-2.09 ± 0.91%) significantly with caffeine compared to control. By 15 minutes in light, DCP VD reversed and was significantly decreased (-5.45 ± 2.62%), whereas MCP VD increased (4.65 ± 1.74%). There were no differences in AFI or VLD. Conclusions We show that, overall, caffeine causes a trend of delayed vascular response in all three macular capillary plexuses in response to ambient light. Whereas the MCP is constricted in the dark, during the transition from dark to light, there is initially delay followed by prolonged constriction of the DCP and constriction followed by slow dilation of the SCP. We posit that these delayed vascular responses may present potential risk of capillary ischemia.

Almost every neuron in the early visual system has some form of an antagonistic receptive field surround. But the function of the surround is poorly understood, especially under naturalistic stimulus conditions. Anatomical and functional characterization of the surround of retinal ganglion cells suggests that surround signals combine with the excitatory feedforward pathway upstream of an important synaptic nonlinearity between bipolar cells and postsynaptic ganglion cells. This circuit architecture suggests at least two unexplored consequences for receptive field structure. First, center and surround inputs should interact nonlinearly, even prior to the summation across visual space that forms the full receptive field center. Second, inputs to the surround may alter the sensitivity of the center to spatial contrast in a scene. We test these hypotheses using both synthetic and naturalistic visual stimuli, and find that the statistics of natural scenes promote these nonlinear interactions. This work shows that, unexpectedly, the surround modulates spatial contrast encoding based on visual context.

Chromatin loops enable transcription factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors, such as active forms of Notch, can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3-dimensional (3D) organization of the cancer genome is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B-cell lymphoma, we show that far beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes through establishing new long-range regulatory interactions. Moreover, a large fraction of Notch-promoted regulatory loops forms highly interacting enhancer and promoter spatial clusters, termed “3D cliques”. Loss-and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML). ITD mutations of Fms-like tyrosine kinase 3 (FLT3) receptor increase kinase activity, and are associated with poor prognostic outcomes. Currently, several small-moleculeFLT3inhibitors (FLT3i) are in clinical trials for targeted therapy of high-risk FLT3-ITD-positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence ofFLT3-ITD mutations in a patient might not guarantee effective clinical response to targeted inhibition ofFLT3kinase. Motivated by the heterogeneity ofFLT3-ITD mutations, we sought to investigate the effects ofFLT3-ITD structural features on response to treatment in AML patients. To this end, we developed HeatITup (HEAT diffusion for Internal Tandem dUPlication), an algorithm to efficiently and accurately identify ITDs and classify them based on their nucleotide composition into newly defined categories of “typical” or “atypical”. Typical ITDs insert sequences are entirely endogenous to theFLT3locus whereas atypical ITDs contain nucleotides exogenous to the wildtypeFLT3. We applied HeatITup to our cohort ofde novoand relapsed AML patients. Individuals with AML carrying typical ITDs benefited significantly more from FLT3i than patients with atypical ITDs, regardless of whether FLT3i was used after initial induction or at relapse. Furthermore, analysis of the TCGA AML cohort demonstrated improved survival for patients with typical ITDs treated with induction chemotherapy. These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing towards personalized treatment for AML patients.

Howard Hughes Medical Institute, Seattle, WA 98195, USASummaryThe distributions of neurons in sensory circuits displayorderedspatialpatternsarrangedtoenhanceorencodespe-cificregionsorfeaturesoftheexternalenvironment.Indeed,visual space is not sampled uniformly across the vertebrateretina. Retinal ganglion cell (RGC) density increases anddendritic arbor size decreases toward retinal locations withhigher sampling frequency, such as the fovea in primatesandareacentralisincarnivores[1].Intheselocations,higheracuity at the level of individual cells is obtained because thereceptive field center of a RGC corresponds approximatelytothespatialextentofitsdendriticarbor[2,3].Formostspe-cies,structurallyandfunctionallydistinctRGCtypesappearto have similar topographies, collectively scaling their celldensities and arbor sizes toward the same retinal location[4]. Thus, visual space is represented across the retina inparallel by multiple distinct circuits [5]. In contrast, we finda population of mouse RGCs, known as alpha or alpha-like[6],thatdisplaysanasal-to-temporalgradientincelldensity,size, and receptive fields, which facilitates enhanced visualsampling in frontal visual fields. The distribution of alpha-like RGCs contrasts with other known mouse RGC typesand suggests that, unlike most mammals, RGC topogra-phies in mice are arranged to sample space differentially.Results and DiscussionMouse ON Alpha-like RGCs Display a Nasal to TemporalGradient in Cell Density and Dendritic Arbor SizeAlpha-like mouse retinal ganglion cells (RGCs) segregateinto three populations: ON-sustained (A

Retinal ganglion cells (RGCs) are frequently divided into functional types by their ability to extract and relay specific features from a visual scene, such as the capacity to discern local or global motion, direction of motion, stimulus orientation, contrast or uniformity, or the presence of large or small objects. Here we introduce three previously uncharacterized, nondirection-selective ON–OFF RGC types that represent a distinct set of feature detectors in the mouse retina. The three high-definition (HD) RGCs possess small receptive-field centers and strong surround suppression. They respond selectively to objects of specific sizes, speeds, and types of motion. We present comprehensive morphological characterization of the HD RGCs and physiological recordings of their light responses, receptive-field size and structure, and synaptic mechanisms of surround suppression. We also explore the similarities and differences between the HD RGCs and a well characterized RGC with a comparably small receptive field, the local edge detector, in response to moving objects and textures. We model populations of each RGC type to study how they differ in their performance tracking a moving object. These results, besides introducing three new RGC types that together constitute a substantial fraction of mouse RGCs, provide insights into the role of different circuits in shaping RGC receptive fields and establish a foundation for continued study of the mechanisms of surround suppression and the neural basis of motion detection.SIGNIFICANCE STATEMENTThe output cells of the retina, retinal ganglion cells (RGCs), are a diverse group of ∼40 distinct neuron types that are often assigned “feature detection” profiles based on the specific aspects of the visual scene to which they respond. Here we describe, for the first time, morphological and physiological characterization of three new RGC types in the mouse retina, substantially augmenting our understanding of feature selectivity. Experiments and modeling show that while these three “high-definition” RGCs share certain receptive-field properties, they also have distinct tuning to the size, speed, and type of motion on the retina, enabling them to occupy different niches in stimulus space.