Genetically encoded barcodes for correlative volume electron microscopy.

While genetically encoded reporters are common for fluorescence microscopy, equivalent multiplexable gene reporters for electron microscopy (EM) are still scarce. Here, by installing a variable number of fixation-stable metal-interacting moieties in the lumen of encapsulin nanocompartments of different sizes, we developed a suite of spherically symmetric and concentric barcodes (EMcapsulins) that are readable by standard EM techniques. Six classes of EMcapsulins could be automatically segmented and differentiated. The coding capacity was further increased by arranging several EMcapsulins into distinct patterns via a set of rigid spacers of variable length. Fluorescent EMcapsulins were expressed to monitor subcellular structures in light and EM. Neuronal expression in Drosophila and mouse brains enabled the automatic identification of genetically defined cells in EM. EMcapsulins are compatible with transmission EM, scanning EM and focused ion beam scanning EM. The expandable palette of genetically controlled EM-readable barcodes can augment anatomical EM images with multiplexed gene expression maps.

A combinatorial code of transcription factors specifies subtypes of visual motion-sensing neurons in Drosophila.

Direction-selective T4/T5 neurons exist in four subtypes, each tuned to visual motion along one of the four cardinal directions. Along with their directional tuning, neurons of each T4/T5 subtype orient their dendrites and project their axons in a subtype-specific manner. Directional tuning, thus, appears strictly linked to morphology in T4/T5 neurons. How the four T4/T5 subtypes acquire their distinct morphologies during development remains largely unknown. Here, we investigated when and how the dendrites of the four T4/T5 subtypes acquire their specific orientations, and profiled the transcriptomes of all T4/T5 neurons during this process. This revealed a simple and stable combinatorial code of transcription factors defining the four T4/T5 subtypes during their development. Changing the combination of transcription factors of specific T4/T5 subtypes resulted in predictable and complete conversions of subtype-specific properties, i.e. dendrite orientation and matching axon projection pattern. Therefore, a combinatorial code of transcription factors coordinates the development of dendrite and axon morphologies to generate anatomical specializations that differentiate subtypes of T4/T5 motion-sensing neurons.

Iron-Sequestering Nanocompartments as Multiplexed Electron Microscopy Gene Reporters.

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

Transcriptional control of morphological properties of direction-selective T4/T5 neurons in Drosophila.

In the Drosophila visual system, T4/T5 neurons represent the first stage of computation of the direction of visual motion. T4 and T5 neurons exist in four subtypes, each responding to motion in one of the four cardinal directions and projecting axons into one of the four lobula plate layers. However, all T4/T5 neurons share properties essential for sensing motion. How T4/T5 neurons acquire their properties during development is poorly understood. We reveal that the transcription factors SoxN and Sox102F control the acquisition of properties common to all T4/T5 neuron subtypes, i.e. the layer specificity of dendrites and axons. Accordingly, adult flies are motion blind after disruption of SoxN or Sox102F in maturing T4/T5 neurons. We further find that the transcription factors Ato and Dac are redundantly required in T4/T5 neuron progenitors for SoxN and Sox102F expression in T4/T5 neurons, linking the transcriptional programmes specifying progenitor identity to those regulating the acquisition of morphological properties in neurons. Our work will help to link structure, function and development in a neuronal type performing a computation that is conserved across vertebrate and invertebrate visual systems.

STED Imaging in Drosophila Brain Slices.

Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy. This protocol allowed us to image small dendritic branches from neurons located deep in the fly brain with improved resolution compared with conventional light microscopy.

Converging axons collectively initiate and maintain synaptic selectivity in a constantly remodeling sensory organ.

Sensory receptors are the functional link between the environment and the brain. The repair of sensory organs enables animals to continuously detect environmental stimuli. However, receptor cell turnover can affect sensory acuity by changing neural connectivity patterns. In zebrafish, two to four postsynaptic lateralis afferent axons converge into individual peripheral mechanosensory organs called neuromasts, which contain hair cell receptors of opposing planar polarity. Yet, each axon exclusively synapses with hair cells of identical polarity during development and regeneration to transmit unidirectional mechanical signals to the brain. The mechanism that governs this exceptionally accurate and resilient synaptic selectivity remains unknown. We show here that converging axons are mutually dependent for polarity-selective connectivity. If rendered solitary, these axons establish simultaneous functional synapses with hair cells of opposing polarities to transmit bidirectional mechanical signals. Remarkably, nonselectivity by solitary axons can be corrected upon the reintroduction of additional axons. Collectively, our results suggest that lateralis synaptogenesis is intrinsically nonselective and that interaxonal interactions continuously rectify mismatched synapses. This dynamic organization of neural connectivity may represent a general solution to maintain coherent synaptic transmission from sensory organs undergoing frequent variations in the number and spatial distribution of receptor cells.

Developmental and architectural principles of the lateral-line neural map.

The transmission and central representation of sensory cues through the accurate construction of neural maps is essential for animals to react to environmental stimuli. Structural diversity of sensorineural maps along a continuum between discrete- and continuous-map architectures can influence behavior. The mechanosensory lateral line of fishes and amphibians, for example, detects complex hydrodynamics occurring around the animal body. It triggers innate fast escape reactions but also modulates complex navigation behaviors that require constant knowledge about the environment. The aim of this article is to summarize recent work in the zebrafish that has shed light on the development and structure of the lateralis neural map, which is helping to understand how individual sensory modalities generate appropriate behavioral responses to the sensory context.

Neuronal birth order identifies a dimorphic sensorineural map.

Spatially distributed sensory information is topographically mapped in the brain by point-to-point correspondence of connections between peripheral receptors and central target neurons. In fishes, for example, the axonal projections from the mechanosensory lateral line organize a somatotopic neural map. The lateral line provides hydrodynamic information for intricate behaviors such as navigation and prey detection. It also mediates fast startle reactions triggered by the Mauthner cell. However, it is not known how the lateralis neural map is built to subserve these contrasting behaviors. Here we reveal that birth order diversifies lateralis afferent neurons in the zebrafish. We demonstrate that early- and late-born lateralis afferents diverge along the main axes of the hindbrain to synapse with hundreds of second-order targets. However, early-born afferents projecting from primary neuromasts also assemble a separate map by converging on the lateral dendrite of the Mauthner cell, whereas projections from secondary neuromasts never make physical contact with the Mauthner cell. We also show that neuronal diversity and map topology occur normally in animals permanently deprived of mechanosensory activity. We conclude that neuronal birth order correlates with the assembly of neural submaps, whose combination is likely to govern appropriate behavioral reactions to the sensory context.

Progressive neurogenesis defines lateralis somatotopy.

Fishes and amphibians localize hydromechanical variations along their bodies using the lateral-line sensory system. This is possible because the spatial distribution of neuromasts is represented in the hindbrain by a somatotopic organization of the lateralis afferent neurons' central projections. The mechanisms that establish lateralis somatotopy are not known. Using BAPTI and neuronal tracing in the zebrafish, we demonstrate growth anisotropy of the posterior lateralis ganglion. We characterized a new transgenic line for in vivo imaging to show that although peripheral growth-cone structure adumbrates somatotopy, the order of neurogenesis represents a more accurate predictor of the position of a neuron's central axon along the somatotopic axis in the hindbrain. We conclude that progressive neurogenesis defines lateralis somatotopy.

Multispectral four-dimensional imaging reveals that evoked activity modulates peripheral arborization and the selection of plane-polarized targets by sensory neurons.

The polarity of apical stereocilia endows hair cells with directional excitability, which in turn enables animals to determine the vectorial component of a sound. Neuromasts of the lateral line of aquatic vertebrates harbor two populations of hair cells that are oriented at 180 degrees relative to each other. The resulting sensory-vectorial ambiguity is solved by lateralis afferent neurons that discriminate between hair cells of opposite polarities to innervate only those with the same orientation. How neurons select identically oriented hair cells remains unknown. To gain insight into the mechanism that underlies this selection, we devised a simple method to gather dynamic morphometric information about axonal terminals in toto by four-dimensional imaging. Applying this strategy to the zebrafish allowed us to correlate hair cell orientation to single afferent neurons at subcellular resolution. Here we show that in zebrafish with absent hair cell mechanoreception, lateralis afferents arborize profusely in the periphery, display less stability, and make improper target selections. Central axons, however, show no dynamic changes and establish normal contacts with the Mauthner cell, a characteristic second-order target in the hindbrain. We propose that the hardwired developmental mechanisms that underlie peripheral arborization and target recognition are modulated by evoked hair cell activity. This interplay between intrinsic and extrinsic cues is essential for plane-polarized target selection by lateralis afferent neurons.

Afferent neurons of the zebrafish lateral line are strict selectors of hair-cell orientation.

Hair cells in the inner ear display a characteristic polarization of their apical stereocilia across the plane of the sensory epithelium. This planar orientation allows coherent transduction of mechanical stimuli because the axis of morphological polarity of the stereocilia corresponds to the direction of excitability of the hair cells. Neuromasts of the lateral line in fishes and amphibians form two intermingled populations of hair cells oriented at 180 degrees relative to each other, however, creating a stimulus-polarity ambiguity. Therefore, it is unknown how these animals resolve the vectorial component of a mechanical stimulus. Using genetic mosaics and live imaging in transgenic zebrafish to visualize hair cells and neurons at single-cell resolution, we show that lateral-line afferents can recognize the planar polarization of hair cells. Each neuron forms synapses with hair cells of identical orientation to divide the neuromast into functional planar-polarity compartments. We also show that afferent neurons are strict selectors of polarity that can re-establish synapses with identically oriented targets during hair-cell regeneration. Our results provide the anatomical bases for the physiological models of signal-polarity resolution by the lateral line.