Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (∼10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, the efficacy of exogenous RNA interference (RNAi) varies dramatically within the Caenorhabditis elegans natural population, raising questions about our understanding of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some wild strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others, the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains but dampens it further in others. Among wild strains, genes known to mediate RNAi exhibited very high expression variation relative to other genes in the genome as well as allelic divergence and strain-specific instances of pseudogenization at the sequence level. Our results demonstrate functional diversification in the small RNA pathways in C. elegans and suggest that RNAi processes are evolving rapidly and dynamically in nature.

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

The C9orf72 hexanucleotide repeat expansion (HRE) is the most frequent genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we describe the pathogenic cascades that are initiated by the C9orf72 HRE DNA. The HRE DNA binds to its protein partner DAXX and promotes its liquid-liquid phase separation, which is capable of reorganizing genomic structures. An HRE-dependent nuclear accumulation of DAXX drives chromatin remodeling and epigenetic changes such as histone hypermethylation and hypoacetylation in patient cells. While regulating global gene expression, DAXX plays a key role in the suppression of basal and stress-inducible expression of C9orf72 via chromatin remodeling and epigenetic modifications of the promoter of the major C9orf72 transcript. Downregulation of DAXX or rebalancing the epigenetic modifications mitigates the stress-induced sensitivity of C9orf72-patient-derived motor neurons. These studies reveal a C9orf72 HRE DNA-dependent regulatory mechanism for both local and genomic architectural changes in the relevant diseases.

Progressive depletion of midbrain dopamine neurons (PDD) is associated with deficits in the initiation, speed, and fluidity of voluntary movement. Models of basal ganglia function focus on initiation deficits; however, it is unclear how they account for deficits in the speed or amplitude of movement (vigor). Using an effort-based operant conditioning task for head-fixed mice, we discovered distinct functional classes of neurons in the dorsal striatum that represent movement vigor. Mice with PDD exhibited a progressive reduction in vigor, along with a selective impairment of its neural representation in striatum. Restoration of dopaminergic tone with a synthetic precursor ameliorated deficits in movement vigor and its neural representation, while suppression of striatal activity during movement was sufficient to reduce vigor. Thus, dopaminergic input to the dorsal striatum is indispensable for the emergence of striatal activity that mediates adaptive changes in movement vigor. These results suggest refined intervention strategies for Parkinson’s disease.

Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. Previously we described the anatomy of the adult MB and defined 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments (Aso et al., 2014a; Aso et al., 2014b). Here we compare the properties of memories formed by optogenetic activation of individual DAN cell types. We found extensive differences in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. Our results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences.

Somatic sexual dimorphisms outside of the nervous system in Drosophila melanogaster are largely controlled by the male- and female-specific Doublesex transcription factors (DSX(M) and DSX(F), respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with doublesex (dsx) gene function, we focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. We show that DSX(M) and DSX(F) promote and repress GSO formation, respectively, and that their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. Our results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSX(F) then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX.

It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The Drosophila melanogaster male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively. We report the discovery of male-specific thoracic interneurons—the TN1A neurons—that are required specifically for sine song. The TN1A neurons can drive the activity of a sex-non-specific wing motoneuron, hg1, which is also required for sine song. The male-specific connection between the TN1A neurons and the hg1 motoneuron is regulated by the sexual differentiation gene doublesex. We find that doublesex is required in the TN1A neurons during development to increase the density of the TN1A arbors that interact with dendrites of the hg1motoneuron. Our findings demonstrate how a sexual differentiation gene can build a sex-specific circuit motif by modulating neuronal arborization.

•Doublesex-expressing TN1 neurons are necessary and sufficient for the male sine song•A subclass of TN1 neurons, TN1A, contributes to the sine song•TN1A neurons are functionally coupled to a sine song motoneuron, hg1•Doublesex regulates the connectivity between the TN1A and hg1 neurons

It is unclear how developmental regulatory genes specify sex-specific behaviors. Shirangi et al. demonstrate that the Drosophila sexual differentiation gene doublesex encodes a sex-specific behavior—male song—by promoting the connectivity between the male-specific TN1A neurons and the sex-non-specific hg1 neurons, which are required for production of the song.

The mushroom body (MB) is the center for associative learning in insects. In Drosophila, intersectional split-GAL4 drivers and electron microscopy (EM) connectomes have laid the foundation for precise interrogation of the MB neural circuits. However, many cell types upstream and downstream of the MB remained to be investigated due to lack of driver lines. Here we describe a new collection of over 800 split-GAL4 and split-LexA drivers that cover approximately 300 cell types, including sugar sensory neurons, putative nociceptive ascending neurons, olfactory and thermo-/hygro-sensory projection neurons, interneurons connected with the MB-extrinsic neurons, and various other cell types. We characterized activation phenotypes for a subset of these lines and identified the sugar sensory neuron line most suitable for reward substitution. Leveraging the thousands of confocal microscopy images associated with the collection, we analyzed neuronal morphological stereotypy and discovered that one set of mushroom body output neurons, MBON08/MBON09, exhibits striking individuality and asymmetry across animals. In conjunction with the EM connectome maps, the driver lines reported here offer a powerful resource for functional dissection of neural circuits for associative learning in adult Drosophila.

We describe a method for molecular confinement and single-fluorophore sensitive measurement in aqueous nanodroplets in oil. The sequestration of individual molecules in droplets has become a useful tool in genomics and molecular evolution. Similarly, the use of single fluorophores, or pairs of fluorophores, to study biomolecular interactions and structural dynamics is now common. Most often these single-fluorophore sensitive measurements are performed on molecules that are surface attached. Confinement via surface attachment permits molecules to be located and studied for a prolonged period of time. For molecules that denature on surfaces, for interactions that are transient or out-of-equilibrium, or to observe the dynamic equilibrium of freely diffusing reagents, surface attachment may not be an option. In these cases, droplet confinement presents an alternative method for molecular confinement. Here, we describe this method as used in single-fluorophore sensitive measurement and discuss its advantages, limitations, and future prospects.