Retinoic Acid-Related Orphan Receptor Beta (RORβ) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORβ is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORβ delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORβ, , is down-regulated without RORβ, and knock-out alone disrupts TCA organization in adult barrels.

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of “glitter bomb” labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

The courtship song of Drosophila melanogaster has long served as an excellent model system for studies of animal communication and differences in courtship song have been demonstrated among populations and between species. Here, we report that flies of African and European origin, which diverged approximately 13,000 years ago, show significant genetic differentiation in the use of slow versus fast pulse song. Using a combination of quantitative trait mapping and population genetic analysis we detected a single strong QTL underlying this trait and we identified candidate genes that may contribute to the evolution of this trait. Song trait variation between parental strains of our recombinant inbred panel enabled detection of genomic intervals associated with six additional song traits, some of which include known courtship-related genes. These findings improve the prospects for further genetic insights into the evolution of reproductive behavior and the biology underlying courtship song.

bioRxiv Preprint: https://www.biorxiv.org/content/early/2024/05/17/2024.05.14.594231

The small size and translucency of larval zebrafish () have made it a unique experimental system to investigate whole-brain neural circuit structure and function. Still, the connectivity patterns between most neuronal types remain mostly unknown. This gap in knowledge underscores the critical need for effective neural circuit mapping tools, especially ones that can integrate structural and functional analyses. To address this, we previously developed a vesicular stomatitis virus (VSV) based approach called Tracer with Restricted Anterograde Spread (TRAS). TRAS utilizes lentivirus to complement replication-incompetent VSV (VSVΔG) to allow restricted (monosynaptic) anterograde labeling from projection neurons to their target cells in the brain. Here, we report the second generation of TRAS (TRAS-M51R), which utilizes a mutant variant of VSVΔG [VSV(M51R)ΔG] with reduced cytotoxicity. Within the primary visual pathway, we found that TRAS-M51R significantly improved long-term viability of transsynaptic labeling (compared to TRAS) while maintaining anterograde spread activity. By using Cre-expressing VSV(M51R)ΔG, TRAS-M51R could selectively label excitatory ( positive) and inhibitory ( positive) retinorecipient neurons. We further show that these labeled excitatory and inhibitory retinorecipient neurons retained neuronal excitability upon visual stimulation at 5-8 days post fertilization (2-5 days post-infection). Together, these findings show that TRAS-M51R is suitable for neural circuit studies that integrate structural connectivity, cell-type identity, and neurophysiology.

Dynamic imaging of genomic loci is key for understanding gene regulation, but methods for imaging genomes, in particular non-repetitive DNAs, are limited. We developed CRISPRdelight, a DNA-labeling system based on endonuclease-deficient CRISPR-Cas12a (dCas12a), with an engineered CRISPR array to track DNA location and motion. CRISPRdelight enables robust imaging of all examined 12 non-repetitive genomic loci in different cell lines. We revealed the confined movement of the CCAT1 locus (chr8q24) at the nuclear periphery for repressed expression and active motion in the interior nucleus for transcription. We uncovered the selective repositioning of HSP gene loci to nuclear speckles, including a remarkable relocation of HSPH1 (chr13q12) for elevated transcription during stresses. Combining CRISPR-dCas12a and RNA aptamers allowed multiplex imaging of four types of satellite DNA loci with a single array, revealing their spatial proximity to the nucleolus-associated domain. CRISPRdelight is a user-friendly and robust system for imaging and tracking genomic dynamics and regulation.

The inner mitochondrial membrane (IMM) is the site of bulk ATP generation in cells and has a broadly conserved lipid composition enriched in unsaturated phospholipids and cardiolipin (CL). While proteins that shape the IMM and its characteristic cristae membranes (CM) have been defined, specific mechanisms by which mitochondrial lipids dictate its structure and function have yet to be elucidated. Here we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions shape CM morphology and ATP generation. When modulating fatty acid unsaturation in engineered yeast strains, we observed that loss of di-unsaturated phospholipids (PLs) led to a breakpoint in IMM topology and respiratory capacity. We found that PL unsaturation modulates the organization of ATP synthases that shape cristae ridges. Based on molecular modeling of mitochondrial-specific membrane adaptations, we hypothesized that conical lipids like CL buffer against the effects of saturation on the IMM. In cells, we discovered that loss of CL collapses the IMM at intermediate levels of PL saturation, an effect that is independent of ATP synthase oligomerization. To explain this interaction, we employed a continuum modeling approach, finding that lipid and protein-mediated curvatures are predicted to act in concert to form curved membranes in the IMM. The model highlighted a snapthrough instability in cristae tubule formation, which could drive IMM collapse upon small changes in composition. The interaction between CL and di-unsaturated PLs suggests that growth conditions that alter the fatty acid pool, such as oxygen availability, could define CL function. While loss of CL only has a minimal phenotype under standard laboratory conditions, we show that its synthesis is essential under microaerobic conditions that better mimic natural yeast fermentation. Lipid and protein-mediated mechanisms of curvature generation can thus act together to support mitochondrial architecture under changing environments.

Animal behavioural diversity ultimately stems from variation in neural circuitry, yet how central neural circuits evolve remains poorly understood. Studies of neural circuit evolution often focus on a few elements within a network. However, addressing fundamental questions in evolutionary neuroscience, such as whether some elements are more evolvable than others, requires a more global and unbiased approach. Here, we used synapse-level comparative connectomics to examine how an entire olfactory circuit evolves. We compared the full antennal lobe connectome of the larvae of two closely related Drosophila species, D. melanogaster and D. erecta, which differ in their ecological niches and odour-driven behaviours. We found that evolutionary change is unevenly distributed across the network. Some features, including neuron types, neuron numbers and interneuron-to-interneuron connectivity, are highly conserved. These conserved elements delineate a core circuit blueprint presumably required for fundamental olfactory processing. Superimposed on this scaffold, we find rewiring changes that mirror each species ecologies, including a systematic shift in the excitation-to-inhibition balance in the feedforward pathways. We further show that some neurons have changed more than others, and that even within individual neurons some synaptic elements remain conserved while others display major species-specific changes, suggesting evolutionary hot-spots within the circuit. Our findings reveal constrained and adaptable elements within olfactory networks, and establish a framework for identifying general principles in the evolution of neural circuits underlying behaviour.

Metabolism is fundamental to organism physiology and pathology. From the intricate network of metabolic reactions, diverse chemical molecules, collectively termed as metabolites, are produced. In multicellular organisms, metabolite communication between different tissues is vital for maintaining homeostasis and adaptation. However, the molecular mechanisms mediating these metabolite communications remain poorly understood. Here, we focus on nucleosides and nucleotides, essential metabolites involved in multiple cellular processes, and report the pivotal role of the SLC29A family of transporters in mediating nucleoside coordination between the soma and the germline. Through genetic analysis, we discovered that two Caenorhabditis elegans homologs of SLC29A transporters, Equilibrative Nucleoside Transporter ENT-1 and ENT-2, act in the germline and the intestine, respectively, to regulate reproduction. Their knockdown synergistically results in sterility. Further single-cell transcriptomic and targeted metabolomic profiling revealed that the ENT double knockdown specifically affects genes in the purine biosynthesis pathway and reduces the ratio of guanosine to adenosine levels. Importantly, guanosine supplementation into the body cavity/pseudocoelom through microinjection rescued the sterility caused by the ENT double knockdown, whereas adenosine microinjection had no effect. Together, these studies support guanosine as a rate limiting factor in the control of reproduction, uncover the previously unknown nucleoside/nucleotide communication between the soma and the germline essential for reproductive success, and highlight the significance of SLC-mediated cell-nonautonomous metabolite coordination in regulating organism physiology.

Preprint: https://doi.org/10.1101/2024.09.12.612591

To stimulate progress in automating the reconstruction of neural circuits, we organized the first international challenge on 2D segmentation of electron microscopic (EM) images of the brain. Participants submitted boundary maps predicted for a test set of images, and were scored based on their agreement with a consensus of human expert annotations. The winning team had no prior experience with EM images, and employed a convolutional network. This “deep learning” approach has since become accepted as a standard for segmentation of EM images. The challenge has continued to accept submissions, and the best so far has resulted from cooperation between two teams. The challenge has probably saturated, as algorithms cannot progress beyond limits set by ambiguities inherent in 2D scoring and the size of the test dataset. Retrospective evaluation of the challenge scoring system reveals that it was not sufficiently robust to variations in the widths of neurite borders. We propose a solution to this problem, which should be useful for a future 3D segmentation challenge.

Cryo-ultramicrotomy, developed by Bernhard in 1965 [1], has long been regarded as the pinnacle of achievement for electron microscopists. This technique allows biological samples to be sliced into ultrathin sections and examined in a cryo-electron microscope, revealing the most intricate subcellular structures without chemical fixation or staining. The advent of vitrification [2,3] and high-pressure freezing (HPF) technology [4,5] provided reliable methods for preserving cellular structures, and the introduction of diamond knife to cryo-ultramicrotomy [6] offering cryo-ultramicrotomists reassurance in consistency of the quality [7].