Automated profiling of gene function during embryonic development.

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

Vision-dependent specification of cell types and function in the developing cortex.

The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.

Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.

Preservation of vision after CaMKII-mediated protection of retinal ganglion cells.

Retinal ganglion cells (RGCs) are the sole output neurons that transmit visual information from the retina to the brain. Diverse insults and pathological states cause degeneration of RGC somas and axons leading to irreversible vision loss. A fundamental question is whether manipulation of a key regulator of RGC survival can protect RGCs from diverse insults and pathological states, and ultimately preserve vision. Here, we report that CaMKII-CREB signaling is compromised after excitotoxic injury to RGC somas or optic nerve injury to RGC axons, and reactivation of this pathway robustly protects RGCs from both injuries. CaMKII activity also promotes RGC survival in the normal retina. Further, reactivation of CaMKII protects RGCs in two glaucoma models where RGCs degenerate from elevated intraocular pressure or genetic deficiency. Last, CaMKII reactivation protects long-distance RGC axon projections in vivo and preserves visual function, from the retina to the visual cortex, and visually guided behavior.

The Visual Opsin Gene Repertoires of Teleost Fishes: Evolution, Ecology, and Function.

Visual opsin genes expressed in the rod and cone photoreceptor cells of the retina are core components of the visual sensory system of vertebrates. Here, we provide an overview of the dynamic evolution of visual opsin genes in the most species-rich group of vertebrates, teleost fishes. The examination of the rich genomic resources now available for this group reveals that fish genomes contain more copies of visual opsin genes than are present in the genomes of amphibians, reptiles, birds, and mammals. The expansion of opsin genes in fishes is due primarily to a combination of ancestral and lineage-specific gene duplications. Following their duplication, the visual opsin genes of fishes repeatedly diversified at the same key spectral-tuning sites, generating arrays of visual pigments sensitive to the ultraviolet to red spectrum of light. Species-specific opsin gene repertoires correlate strongly with underwater light habitats, ecology, and color-based sexual selection.

Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoantennae.

Mammals cannot see light over 700 nm in wavelength. This limitation is due to the physical thermodynamic properties of the photon-detecting opsins. However, the detection of naturally invisible near-infrared (NIR) light is a desirable ability. To break this limitation, we developed ocular injectable photoreceptor-binding upconversion nanoparticles (pbUCNPs). These nanoparticles anchored on retinal photoreceptors as miniature NIR light transducers to create NIR light image vision with negligible side effects. Based on single-photoreceptor recordings, electroretinograms, cortical recordings, and visual behavioral tests, we demonstrated that mice with these nanoantennae could not only perceive NIR light, but also see NIR light patterns. Excitingly, the injected mice were also able to differentiate sophisticated NIR shape patterns. Moreover, the NIR light pattern vision was ambient-daylight compatible and existed in parallel with native daylight vision. This new method will provide unmatched opportunities for a wide variety of emerging bio-integrated nanodevice designs and applications. VIDEO ABSTRACT.

Visual Projection Neurons Mediating Directed Courtship in Drosophila.

Many animals rely on vision to detect, locate, and track moving objects. In Drosophila courtship, males primarily use visual cues to orient toward and follow females and to select the ipsilateral wing for courtship song. Here, we show that the LC10 visual projection neurons convey essential visual information during courtship. Males with LC10 neurons silenced are unable to orient toward or maintain proximity to the female and do not predominantly use the ipsilateral wing when singing. LC10 neurons preferentially respond to small moving objects using an antagonistic motion-based center-surround mechanism. Unilateral activation of LC10 neurons recapitulates the orienting and ipsilateral wing extension normally elicited by females, and the potency with which LC10 induces wing extension is enhanced in a state of courtship arousal controlled by male-specific P1 neurons. These data suggest that LC10 is a major pathway relaying visual input to the courtship circuits in the male brain.

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire.

Color Processing in the Early Visual System of Drosophila.

Color vision extracts spectral information by comparing signals from photoreceptors with different visual pigments. Such comparisons are encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. Here, we examine the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. We report that color-opponent processing of UVshort/blue and UVlong/green is already implemented in R7/R8 inner photoreceptor terminals of "pale" and "yellow" ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently.

The Code for Facial Identity in the Primate Brain.

Primates recognize complex objects such as faces with remarkable speed and reliability. Here, we reveal the brain's code for facial identity. Experiments in macaques demonstrate an extraordinarily simple transformation between faces and responses of cells in face patches. By formatting faces as points in a high-dimensional linear space, we discovered that each face cell's firing rate is proportional to the projection of an incoming face stimulus onto a single axis in this space, allowing a face cell ensemble to encode the location of any face in the space. Using this code, we could precisely decode faces from neural population responses and predict neural firing rates to faces. Furthermore, this code disavows the long-standing assumption that face cells encode specific facial identities, confirmed by engineering faces with drastically different appearance that elicited identical responses in single face cells. Our work suggests that other objects could be encoded by analogous metric coordinate systems. PAPERCLIP.

Mapping the Neural Substrates of Behavior.

Assigning behavioral functions to neural structures has long been a central goal in neuroscience and is a necessary first step toward a circuit-level understanding of how the brain generates behavior. Here, we map the neural substrates of locomotion and social behaviors for Drosophila melanogaster using automated machine-vision and machine-learning techniques. From videos of 400,000 flies, we quantified the behavioral effects of activating 2,204 genetically targeted populations of neurons. We combined a novel quantification of anatomy with our behavioral analysis to create brain-behavior correlation maps, which are shared as browsable web pages and interactive software. Based on these maps, we generated hypotheses of regions of the brain causally related to sensory processing, locomotor control, courtship, aggression, and sleep. Our maps directly specify genetic tools to target these regions, which we used to identify a small population of neurons with a role in the control of walking.

Quantitative Predictions Orchestrate Visual Signaling in Drosophila.

Vision influences behavior, but ongoing behavior also modulates vision in animals ranging from insects to primates. The function and biophysical mechanisms of most such modulations remain unresolved. Here, we combine behavioral genetics, electrophysiology, and high-speed videography to advance a function for behavioral modulations of visual processing in Drosophila. We argue that a set of motion-sensitive visual neurons regulate gaze-stabilizing head movements. We describe how, during flight turns, Drosophila perform a set of head movements that require silencing their gaze-stability reflexes along the primary rotation axis of the turn. Consistent with this behavioral requirement, we find pervasive motor-related inputs to the visual neurons, which quantitatively silence their predicted visual responses to rotations around the relevant axis while preserving sensitivity around other axes. This work proposes a function for a behavioral modulation of visual processing and illustrates how the brain can remove one sensory signal from a circuit carrying multiple related signals.

Cortical Integration of Vestibular and Visual Cues for Navigation, Visual Processing, and Perception.

Despite increasing evidence of its involvement in several key functions of the cerebral cortex, the vestibular sense rarely enters our consciousness. Indeed, the extent to which these internal signals are incorporated within cortical sensory representation and how they might be relied upon for sensory-driven decision-making, during, for example, spatial navigation, is yet to be understood. Recent novel experimental approaches in rodents have probed both the physiological and behavioral significance of vestibular signals and indicate that their widespread integration with vision improves both the cortical representation and perceptual accuracy of self-motion and orientation. Here, we summarize these recent findings with a focus on cortical circuits involved in visual perception and spatial navigation and highlight the major remaining knowledge gaps. We suggest that vestibulo-visual integration reflects a process of constant updating regarding the status of self-motion, and access to such information by the cortex is used for sensory perception and predictions that may be implemented for rapid, navigation-related decision-making.

Epigenome-metabolism nexus in the retina: implications for aging and disease.

Intimate links between epigenome modifications and metabolites allude to a crucial role of cellular metabolism in transcriptional regulation. Retina, being a highly metabolic tissue, adapts by integrating inputs from genetic, epigenetic, and extracellular signals. Precise global epigenomic signatures guide development and homeostasis of the intricate retinal structure and function. Epigenomic and metabolic realignment are hallmarks of aging and highlight a link of the epigenome-metabolism nexus with aging-associated multifactorial traits affecting the retina, including age-related macular degeneration and glaucoma. Here, we focus on emerging principles of epigenomic and metabolic control of retinal gene regulation, with emphasis on their contribution to human disease. In addition, we discuss potential mitigation strategies involving lifestyle changes that target the epigenome-metabolome relationship for maintaining retinal function.

Cardiomyocyte orientation recovery at micrometer scale reveals long-axis fiber continuum in heart walls.

Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.

Probing Computation in the Primate Visual System at Single-Cone Resolution.

Daylight vision begins when light activates cone photoreceptors in the retina, creating spatial patterns of neural activity. These cone signals are then combined and processed in downstream neural circuits, ultimately producing visual perception. Recent technical advances have made it possible to deliver visual stimuli to the retina that probe this processing by the visual system at its elementary resolution of individual cones. Physiological recordings from nonhuman primate retinas reveal the spatial organization of cone signals in retinal ganglion cells, including how signals from cones of different types are combined to support both spatial and color vision. Psychophysical experiments with human subjects characterize the visual sensations evoked by stimulating a single cone, including the perception of color. Future combined physiological and psychophysical experiments focusing on probing the elementary visual inputs are likely to clarify how neural processing generates our perception of the visual world.

CXCL12 promotes the crossing of retinal ganglion cell axons at the optic chiasm.

Binocular vision requires the segregation of retinal ganglion cell (RGC) axons extending from the retina into the ipsilateral and contralateral optic tracts. RGC axon segregation occurs at the optic chiasm, which forms at the ventral diencephalon midline. Using expression analyses, retinal explants and genetically modified mice, we demonstrate that CXCL12 (SDF1) is required for axon segregation at the optic chiasm. CXCL12 is expressed by the meninges bordering the optic pathway, and CXCR4 by both ipsilaterally and contralaterally projecting RGCs. CXCL12 or ventral diencephalon meninges potently promoted axon outgrowth from both ipsilaterally and contralaterally projecting RGCs. Further, a higher proportion of axons projected ipsilaterally in mice lacking CXCL12 or its receptor CXCR4 compared with wild-type mice as a result of misrouting of presumptive contralaterally specified RGC axons. Although RGCs also expressed the alternative CXCL12 receptor ACKR3, the optic chiasm developed normally in mice lacking ACKR3. Our data support a model whereby meningeal-derived CXCL12 helps drive axon growth from CXCR4-expressing RGCs towards the diencephalon midline, enabling contralateral axon growth. These findings further our understanding of the molecular and cellular mechanisms controlling optic pathway development.

Eye blinks as a visual processing stage.

Humans blink their eyes frequently during normal viewing, more often than it seems necessary for keeping the cornea well lubricated. Since the closure of the eyelid disrupts the image on the retina, eye blinks are commonly assumed to be detrimental to visual processing. However, blinks also provide luminance transients rich in spatial information to neural pathways highly sensitive to temporal changes. Here, we report that the luminance modulations from blinks enhance visual sensitivity. By coupling high-resolution eye tracking in human observers with modeling of blink transients and spectral analysis of visual input signals, we show that blinking increases the power of retinal stimulation and that this effect significantly enhances visibility despite the time lost in exposure to the external scene. We further show that, as predicted from the spectral content of input signals, this enhancement is selective for stimuli at low spatial frequencies and occurs irrespective of whether the luminance transients are actively generated or passively experienced. These findings indicate that, like eye movements, blinking acts as a computational component of a visual processing strategy that uses motor behavior to reformat spatial information into the temporal domain.

How small changes to one eye's retinal image can transform the perceived shape of a very familiar object.

Vision can provide useful cues about the geometric properties of an object, like its size, distance, pose, and shape. But how the brain merges these properties into a complete sensory representation of a three-dimensional object is poorly understood. To address this gap, we investigated a visual illusion in which humans misperceive the shape of an object due to a small change in one eye's retinal image. We first show that this illusion affects percepts of a highly familiar object under completely natural viewing conditions. Specifically, people perceived their own rectangular mobile phone to have a trapezoidal shape. We then investigate the perceptual underpinnings of this illusion by asking people to report both the perceived shape and pose of controlled stimuli. Our results suggest that the shape illusion results from distorted cues to object pose. In addition to yielding insights into object perception, this work informs our understanding of how the brain combines information from multiple visual cues in natural settings. The shape illusion can occur when people wear everyday prescription spectacles; thus, these findings also provide insight into the cue combination challenges that some spectacle wearers experience on a regular basis.

A computational account of transsaccadic attentional allocation based on visual gain fields.

Coordination of goal-directed behavior depends on the brain's ability to recover the locations of relevant objects in the world. In humans, the visual system encodes the spatial organization of sensory inputs, but neurons in early visual areas map objects according to their retinal positions, rather than where they are in the world. How the brain computes world-referenced spatial information across eye movements has been widely researched and debated. Here, we tested whether shifts of covert attention are sufficiently precise in space and time to track an object's real-world location across eye movements. We found that observers' attentional selectivity is remarkably precise and is barely perturbed by the execution of saccades. Inspired by recent neurophysiological discoveries, we developed an observer model that rapidly estimates the real-world locations of objects and allocates attention within this reference frame. The model recapitulates the human data and provides a parsimonious explanation for previously reported phenomena in which observers allocate attention to task-irrelevant locations across eye movements. Our findings reveal that visual attention operates in real-world coordinates, which can be computed rapidly at the earliest stages of cortical processing.