A microRNA that controls the emergence of embryonic movement.

Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here, we investigate the genetic basis underlying the embryonic onset of movement in Drosophila focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, we first develop a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos, and using this system, discover that the Drosophila miRNA miR-2b-1 plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging we define that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests we identify a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene - which we term Movement Modulator (Motor) - as a genetic target that mechanistically links miR-2b-1 to the onset of movement. Cell-specific genetic reconstitution of miR-2b-1 expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that miR-2b-1 affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. Our work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in Drosophila as well as in other species.

The vertebrate retina: a window into the evolution of computation in the brain.

Animal brains are probably the most complex computational machines on our planet, and like everything in biology, they are the product of evolution. Advances in developmental and palaeobiology have been expanding our general understanding of how nervous systems can change at a molecular and structural level. However, how these changes translate into altered function - that is, into 'computation' - remains comparatively sparsely explored. What, concretely, does it mean for neuronal computation when neurons change their morphology and connectivity, when new neurons appear or old ones disappear, or when transmitter systems are slowly modified over many generations? And how does evolution use these many possible knobs and dials to constantly tune computation to give rise to the amazing diversity in animal behaviours we see today? Addressing these major gaps of understanding benefits from choosing a suitable model system. Here, I present the vertebrate retina as one perhaps unusually promising candidate. The retina is ancient and displays highly conserved core organisational principles across the entire vertebrate lineage, alongside a myriad of adjustments across extant species that were shaped by the history of their visual ecology. Moreover, the computational logic of the retina is readily interrogated experimentally, and our existing understanding of retinal circuits in a handful of species can serve as an anchor when exploring the visual circuit adaptations across the entire vertebrate tree of life, from fish deep in the aphotic zone of the oceans to eagles soaring high up in the sky.

Ancient origin of the rod bipolar cell pathway in the vertebrate retina.

Vertebrates rely on rod photoreceptors for vision in low-light conditions. The specialized downstream circuit for rod signalling, called the primary rod pathway, is well characterized in mammals, but circuitry for rod signalling in non-mammals is largely unknown. Here we demonstrate that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA sequencing, we identified two bipolar cell types in zebrafish that are related to mammalian rod bipolar cell (RBCs), the only bipolar type that directly carries rod signals from the outer to the inner retina in the primary rod pathway. By combining electrophysiology, histology and ultrastructural reconstruction of the zebrafish RBCs, we found that, similar to mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells postsynaptic to one RBC type is strikingly similar to that of mammalian RBCs and their amacrine partners, suggesting that the cell types and circuit design of the primary rod pathway emerged before the divergence of teleost fish and mammals. The second RBC type, which forms separate pathways, was either lost in mammals or emerged in fish.

Correction: From water to land: Evolution of photoreceptor circuits for vision in air.

[This corrects the article DOI: 10.1371/journal.pbio.3002422.].

New twists in the evolution of retinal direction selectivity.

In mammals, starburst amacrine cells are centrally involved in motion vision and a new study in PLOS Biology, by Yan and colleagues finds that zebrafish have them, too. They coexist with a second pair of starburst-like neurons, but neither appears to be strongly motion selective.

From water to land: Evolution of photoreceptor circuits for vision in air.

When vertebrates first conquered the land, they encountered a visual world that was radically distinct from that of their aquatic ancestors. Fish exploit the strong wavelength-dependent interactions of light with water by differentially feeding the signals from up to 5 spectral photoreceptor types into distinct behavioural programmes. However, above the water the same spectral rules do not apply, and this called for an update to visual circuit strategies. Early tetrapods soon evolved the double cone, a still poorly understood pair of new photoreceptors that brought the "ancestral terrestrial" complement from 5 to 7. Subsequent nonmammalian lineages differentially adapted this highly parallelised retinal input strategy for their diverse visual ecologies. By contrast, mammals shed most ancestral photoreceptors and converged on an input strategy that is exceptionally general. In eutherian mammals including in humans, parallelisation emerges gradually as the visual signal traverses the layers of the retina and into the brain.

Ancestral photoreceptor diversity as the basis of visual behaviour.

Animal colour vision is based on comparing signals from different photoreceptors. It is generally assumed that processing different spectral types of photoreceptor mainly serves colour vision. Here I propose instead that photoreceptors are parallel feature channels that differentially support visual-motor programmes like motion vision behaviours, prey capture and predator evasion. Colour vision may have emerged as a secondary benefit of these circuits, which originally helped aquatic vertebrates to visually navigate and segment their underwater world. Specifically, I suggest that ancestral vertebrate vision was built around three main systems, including a high-resolution general purpose greyscale system based on ancestral red cones and rods to mediate visual body stabilization and navigation, a high-sensitivity specialized foreground system based on ancestral ultraviolet cones to mediate threat detection and prey capture, and a net-suppressive system based on ancestral green and blue cones for regulating red/rod and ultraviolet circuits. This ancestral strategy probably still underpins vision today, and different vertebrate lineages have since adapted their original photoreceptor circuits to suit their diverse visual ecologies.

Ancient origin of the rod bipolar cell pathway in the vertebrate retina.

Vertebrates rely on rod photoreceptors for vision in low-light conditions. Mammals have a specialized downstream circuit for rod signaling called the primary rod pathway, which comprises specific cell types and wiring patterns that are thought to be unique to this lineage. Thus, it has been long assumed that the primary rod pathway evolved in mammals. Here, we challenge this view by demonstrating that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA-sequencing, we identified two bipolar cell (BC) types in zebrafish that are related to mammalian rod BCs (RBCs) of the primary rod pathway. By combining electrophysiology, histology, and ultrastructural reconstruction of the zebrafish RBCs, we found that, like mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory, and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells post-synaptic to one RBC type is strikingly similar to that of mammalian RBCs, suggesting that the cell types and circuit design of the primary rod pathway have emerged before the divergence of teleost fish and amniotes. The second RBC type, which forms separate pathways, is either lost in mammals or emerged in fish.

Ancient origin of the rod bipolar cell pathway in the vertebrate retina.

Vertebrates rely on rod photoreceptors for vision in low-light conditions1. Mammals have a specialized downstream circuit for rod signaling called the primary rod pathway, which comprises specific cell types and wiring patterns that are thought to be unique to this lineage2-6. Thus, it has been long assumed that the primary rod pathway evolved in mammals3,5-7. Here, we challenge this view by demonstrating that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA-sequencing, we identified two bipolar cell (BC) types in zebrafish that are related to mammalian rod BCs (RBCs) of the primary rod pathway. By combining electrophysiology, histology, and ultrastructural reconstruction of the zebrafish RBCs, we found that, like mammalian RBCs8, both zebrafish RBC types connect with all rods and red-cones in their dendritic territory, and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells post-synaptic to one RBC type is strikingly similar to that of mammalian RBCs. This suggests that the cell types and circuit design of the primary rod pathway may have emerged before the divergence of teleost fish and amniotes (mammals, bird, reptiles). The second RBC type in zebrafish, which forms separate pathways from the first RBC type, is either lost in mammals or emerged in fish to serve yet unknown roles.

Birds multiplex spectral and temporal visual information via retinal On- and Off-channels.

In vertebrate vision, early retinal circuits divide incoming visual information into functionally opposite elementary signals: On and Off, transient and sustained, chromatic and achromatic. Together these signals can yield an efficient representation of the scene for transmission to the brain via the optic nerve. However, this long-standing interpretation of retinal function is based on mammals, and it is unclear whether this functional arrangement is common to all vertebrates. Here we show that male poultry chicks use a fundamentally different strategy to communicate information from the eye to the brain. Rather than using functionally opposite pairs of retinal output channels, chicks encode the polarity, timing, and spectral composition of visual stimuli in a highly correlated manner: fast achromatic information is encoded by Off-circuits, and slow chromatic information overwhelmingly by On-circuits. Moreover, most retinal output channels combine On- and Off-circuits to simultaneously encode, or multiplex, both achromatic and chromatic information. Our results from birds conform to evidence from fish, amphibians, and reptiles which retain the full ancestral complement of four spectral types of cone photoreceptors.

Cephalopod-omics: Emerging Fields and Technologies in Cephalopod Biology.

Few animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving field.

Amacrine cells differentially balance zebrafish color circuits in the central and peripheral retina.

The vertebrate inner retina is driven by photoreceptors whose outputs are already pre-processed; in zebrafish, outer retinal circuits split "color" from "grayscale" information across four cone-photoreceptor types. It remains unclear how the inner retina processes incoming spectral information while also combining cone signals to shape grayscale functions. We address this question by imaging the light-driven responses of amacrine cells (ACs) and bipolar cells (BCs) in larval zebrafish in the presence and pharmacological absence of inner retinal inhibition. We find that ACs enhance opponency in some bipolar cells while at the same time suppressing pre-existing opponency in others, so that, depending on the retinal region, the net change in the number of color-opponent units is essentially zero. To achieve this "dynamic balance," ACs counteract intrinsic color opponency of BCs via the On channel. Consistent with these observations, Off-stratifying ACs are exclusively achromatic, while all color-opponent ACs stratify in the On sublamina.

Center-surround interactions underlie bipolar cell motion sensitivity in the mouse retina.

Motion sensing is a critical aspect of vision. We studied the representation of motion in mouse retinal bipolar cells and found that some bipolar cells are radially direction selective, preferring the origin of small object motion trajectories. Using a glutamate sensor, we directly observed bipolar cells synaptic output and found that there are radial direction selective and non-selective bipolar cell types, the majority being selective, and that radial direction selectivity relies on properties of the center-surround receptive field. We used these bipolar cell receptive fields along with connectomics to design biophysical models of downstream cells. The models and additional experiments demonstrated that bipolar cells pass radial direction selective excitation to starburst amacrine cells, which contributes to their directional tuning. As bipolar cells provide excitation to most amacrine and ganglion cells, their radial direction selectivity may contribute to motion processing throughout the visual system.

Is our retina really upside down?

In the vertebrate eye, photoreceptors are covered beneath a thick sheet of neural retina and face away from the light. This seemingly awkward arrangement has led to the popular notion that our retinas are upside down, implying a deep design flaw. Baden and Nilsson argue that, from an evolutionary perspective, an inverted design actually offers many notable benefits that might have never been exploited if things had started off the other way round.

Ancestral circuits for vertebrate color vision emerge at the first retinal synapse.

For color vision, retinal circuits separate information about intensity and wavelength. In vertebrates that use the full complement of four "ancestral" cone types, the nature and implementation of this computation remain poorly understood. Here, we establish the complete circuit architecture of outer retinal circuits underlying color processing in larval zebrafish. We find that the synaptic outputs of red and green cones efficiently rotate the encoding of natural daylight in a principal components analysis­like manner to yield primary achromatic and spectrally opponent axes, respectively. Blue cones are tuned to capture most remaining variance when opposed to green cones, while UV cone present a UV achromatic axis for prey capture. We note that fruitflies use essentially the same strategy. Therefore, rotating color space into primary achromatic and chromatic axes at the eye's first synapse may thus be a fundamental principle of color vision when using more than two spectrally well-separated photoreceptor types.

Spectral inference reveals principal cone-integration rules of the zebrafish inner retina.

Retinal bipolar cells integrate cone signals at dendritic and axonal sites. The axonal route, involving amacrine cells, remains largely uncharted. However, because cone types differ in their spectral sensitivities, insights into bipolar cells' cone integration might be gained based on their spectral tunings. We therefore recorded in vivo responses of bipolar cell presynaptic terminals in larval zebrafish to widefield but spectrally resolved flashes of light and mapped the results onto spectral responses of the four cones. This "spectral circuit mapping" allowed explaining ∼95% of the spectral and temporal variance of bipolar cell responses in a simple linear model, thereby revealing several notable integration rules of the inner retina. Bipolar cells were dominated by red-cone inputs, often alongside equal sign inputs from blue and green cones. In contrast, UV-cone inputs were uncorrelated with those of the remaining cones. This led to a new axis of spectral opponency where red-, green-, and blue-cone "Off" circuits connect to "natively-On" UV-cone circuits in the outermost fraction of the inner plexiform layer-much as how key color opponent circuits are established in mammals. Beyond this, and despite substantial temporal diversity that was not present in the cones, bipolar cell spectral tunings were surprisingly simple. They either approximately resembled both opponent and non-opponent spectral motifs already present in the cones or exhibited a stereotyped non-opponent broadband response. In this way, bipolar cells not only preserved the efficient spectral representations in the cones but also diversified them to set up a total of six dominant spectral motifs, which included three axes of spectral opponency.

Distinct synaptic transfer functions in same-type photoreceptors.

Many sensory systems use ribbon-type synapses to transmit their signals to downstream circuits. The properties of this synaptic transfer fundamentally dictate which aspects in the original stimulus will be accentuated or suppressed, thereby partially defining the detection limits of the circuit. Accordingly, sensory neurons have evolved a wide variety of ribbon geometries and vesicle pool properties to best support their diverse functional requirements. However, the need for diverse synaptic functions does not only arise across neuron types, but also within. Here we show that UV-cones, a single type of photoreceptor of the larval zebrafish eye, exhibit striking differences in their synaptic ultrastructure and consequent calcium to glutamate transfer function depending on their location in the eye. We arrive at this conclusion by combining serial section electron microscopy and simultaneous 'dual-colour' two-photon imaging of calcium and glutamate signals from the same synapse in vivo. We further use the functional dataset to fit a cascade-like model of the ribbon synapse with different vesicle pool sizes, transfer rates, and other synaptic properties. Exploiting recent developments in simulation-based inference, we obtain full posterior estimates for the parameters and compare these across different retinal regions. The model enables us to extrapolate to new stimuli and to systematically investigate different response behaviours of various ribbon configurations. We also provide an interactive, easy-to-use version of this model as an online tool. Overall, we show that already on the synaptic level of single-neuron types there exist highly specialised mechanisms which are advantageous for the encoding of different visual features.

Spherical arena reveals optokinetic response tuning to stimulus location, size, and frequency across entire visual field of larval zebrafish.

Many animals have large visual fields, and sensory circuits may sample those regions of visual space most relevant to behaviours such as gaze stabilisation and hunting. Despite this, relatively small displays are often used in vision neuroscience. To sample stimulus locations across most of the visual field, we built a spherical stimulus arena with 14,848 independently controllable LEDs. We measured the optokinetic response gain of immobilised zebrafish larvae to stimuli of different steradian size and visual field locations. We find that the two eyes are less yoked than previously thought and that spatial frequency tuning is similar across visual field positions. However, zebrafish react most strongly to lateral, nearly equatorial stimuli, consistent with previously reported spatial densities of red, green, and blue photoreceptors. Upside-down experiments suggest further extra-retinal processing. Our results demonstrate that motion vision circuits in zebrafish are anisotropic, and preferentially monitor areas with putative behavioural relevance.

Circuit mechanisms for colour vision in zebrafish.

The use of spectral information in natural light to inform behaviour is one of the oldest and most fundamental abilities of visual systems. It long-predates animals' venture onto the land, and even the appearance of image-forming eyes. Accordingly, circuits for colour vision evolved under the surface of ancient oceans for hundreds of millions of years. These aquatic beginnings fundamentally underpin, and likely constrain, the organisation of modern visual systems. In contrast to our detailed circuit level understanding from diverse terrestrial vertebrates, however, comparatively little is known about their aquatic counterparts. Here, I summarise some of what is known about neural circuits for colour vision in fish, the most species-diverse group of vertebrates. With a focus on zebrafish, I will explore how their computational strategies are linked to the statistics of natural light in the underwater world, and how their study might help us understand vision in general, including in our own eyes.

Colourfulness as a possible measure of object proximity in the larval zebrafish brain.

The encoding of light increments and decrements by separate On- and Off- systems is a fundamental ingredient of vision, which supports edge detection and makes efficient use of the limited dynamic range of visual neurons1. Theory predicts that the neural representation of On- and Off-signals should be balanced, including across an animal's visible spectrum. Here we find that larval zebrafish violate this textbook expectation: in the zebrafish brain, UV-stimulation near exclusively gives On-responses, blue/green stimulation mostly Off-responses, and red-light alone elicits approximately balanced On- and Off-responses (see also references2-4). We link these findings to zebrafish visual ecology, and suggest that the observed spectral tuning boosts the encoding of object 'colourfulness', which correlates with object proximity in their underwater world5.