High-Throughput, Label-Free Detection of DNA Origami in Single-Cell Suspensions Using origamiFISH-Flow.

Structural DNA nanotechnology enables custom fabrication of nanoscale devices and promises diverse biological applications. However, the effects of design on DNA nanostructure (DN)-cell interactions in vitro and in vivo are not yet well-characterized. origamiFISH is a recently developed technique for imaging DNs in cells and tissues. Compared to the use of fluorescent tags, origamiFISH offers label-free and structure-agnostic detection of DNs with significantly improved sensitivity. Here, the origamiFISH technique is extended to quantify DNs in single-cell suspensions, including in nonadherent cells such as subsets of immune cells, via readout by flow cytometry. This method, referred to as origamiFISH-Flow, is high-throughput (e.g., 10 000 cells per second) and compatible with immunostaining for concurrent cell-type and cell-state characterization. It is shown that origamiFISH-Flow provides 20-fold higher signal-to-noise ratio for DN detection compared to dye labeling approaches, leading to the capture of >25-fold more DN+ cells under single-picomolar DN uptake concentrations. Additionally, the use of origamiFISH-Flow is validated to profile the uptake of various DN shapes across multiple cell lines and splenocytes, as well as to quantify in vivo DN accumulation in lymphoid organs. Together, origamiFISH-Flow offers a new tool to interrogate DN interactions with cells and tissues, while providing insights for tailoring their designs in bio-applications.

Universal, label-free, single-molecule visualization of DNA origami nanodevices across biological samples using origamiFISH.

Structural DNA nanotechnology enables the fabrication of user-defined DNA origami nanostructures (DNs) for biological applications. However, the role of DN design during cellular interactions and subsequent biodistribution remain poorly understood. Current methods for tracking DN fates in situ, including fluorescent-dye labelling, suffer from low sensitivity and dye-induced artifacts. Here we present origamiFISH, a label-free and universal method for the single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. origamiFISH targets pan-DN scaffold sequences with hybridization chain reaction probes to achieve 1,000-fold signal amplification. We identify cell-type- and DN shape-specific spatiotemporal distribution patterns within a minute of uptake and at picomolar DN concentrations, 10,000× lower than field standards. We additionally optimize compatibility with immunofluorescence and tissue clearing to visualize DN distribution within tissue cryo-/vibratome sections, slice cultures and whole-mount organoids. Together, origamiFISH enables the accurate mapping of DN distribution across subcellular and tissue barriers for guiding the development of DN-based therapeutics.

The Role of Protocadherin γ in Adult Sensory Neurons and Skin Reinnervation.

The clustered protocadherins (cPcdhs) play a critical role in the patterning of several CNS axon and dendritic arbors, through regulation of homophilic self and neighboring interactions. While not explored, primary peripheral sensory afferents that innervate the epidermis may require similar constraints to convey spatial signals with appropriate fidelity. Here, we show that members of the γ-Pcdh (Pcdhγ) family are expressed in both adult sensory neuron axons and in neighboring keratinocytes that have close interactions during skin reinnervation. Adult mice of both sexes were studied. Pcdhγ knock-down either through small interfering RNA (siRNA) transduction or AAV-Cre recombinase transfection of adult mouse primary sensory neurons from floxed Pcdhγ mice was associated with a remarkable rise in neurite outgrowth and branching. Rises in outgrowth were abrogated by Rac1 inhibition. Moreover, AAV-Cre knock-down in Pcdhγ floxed neurons generated a rise in neurite self-intersections, and a robust rise in neighbor intersections or tiling, suggesting a role in sensory axon repulsion. Interestingly, preconditioned (3-d axotomy) neurons with enhanced growth had temporary declines in Pcdhγ and lessened outgrowth from Pcdhγ siRNA. In vivo, mice with local hindpaw skin Pcdhγ knock-down by siRNA had accelerated reinnervation by new epidermal axons with greater terminal branching and reduced intra-axonal spacing. Pcdhγ knock-down also had reciprocal impacts on keratinocyte density and nuclear size. Taken together, this work provides evidence for a role of Pcdhγ in attenuating outgrowth of sensory axons and their interactions, with implications in how new reinnervating axons following injury fare amid skin keratinocytes that also express Pcdhγ.SIGNIFICANCE STATEMENT The molecular mechanisms and potential constraints that govern skin reinnervation and patterning by sensory axons are largely unexplored. Here, we show that γ-protocadherins (Pcdhγ) may help to dictate interaction not only among axons but also between axons and keratinocytes as the former re-enter the skin during reinnervation. Pcdhγ neuronal knock-down enhances outgrowth in peripheral sensory neurons, involving the growth cone protein Rac1 whereas skin Pcdhγ knock-down generates rises in terminal epidermal axon growth and branching during re-innervation. Manipulation of sensory axon regrowth within the epidermis offers an opportunity to influence regenerative outcomes following nerve injury.

PV-IRES-Cre mouse line targets excitatory granule neurons in the cerebellum.

Parvalbumin-expressing inhibitory neurons (PV-INs) are critical for the balance and fine-tuning of complex neuronal circuits. Studies of PV-IN biology require tools for their specific labeling, targeting and manipulation. Among these, the Cre/LoxP system is the most popular in mice, with the two commonly used PV-Cre lines cited over 5600 times. Here we report in the mouse cerebellar cortex that PV-Cre activity is not restricted to inhibitory neurons. Imaging of Cre-activated reporters demonstrated recombination in excitatory granule cells. We present evidence that PV-Cre recombination is: (1) spatially regulated and lobule specific; (2) detected in granule cells in the external and internal granule cell layers arising from strong, but transient Pvalb expression in progenitors between E13-E15; and (3) delayed in a subset of inhibitory interneurons, asynchronous with PV protein expression. Together, our findings establish the spatio-temporal patterns PV-Cre activation in the mouse cerebellum, raising considerations for conditional targeting of Pvalb-expressing inhibitory populations.

Morphological pseudotime ordering and fate mapping reveal diversification of cerebellar inhibitory interneurons.

Understanding how diverse neurons are assembled into circuits requires a framework for describing cell types and their developmental trajectories. Here we combine genetic fate-mapping, pseudotemporal profiling of morphogenesis, and dual morphology and RNA labeling to resolve the diversification of mouse cerebellar inhibitory interneurons. Molecular layer interneurons (MLIs) derive from a common progenitor population but comprise diverse dendritic-, somatic-, and axon initial segment-targeting interneurons. Using quantitative morphology from 79 mature MLIs, we identify two discrete morphological types and presence of extensive within-class heterogeneity. Pseudotime trajectory inference using 732 developmental morphologies indicate the emergence of distinct MLI types during migration, before reaching their final positions. By comparing MLI identities from morphological and transcriptomic signatures, we demonstrate the dissociation between these modalities and that subtype divergence can be resolved from axonal morphogenesis prior to marker gene expression. Our study illustrates the utility of applying single-cell methods to quantify morphology for defining neuronal diversification.

Engineering DNA Nanostructures to Manipulate Immune Receptor Signaling and Immune Cell Fates.

Immune cells sense, communicate, and logically integrate a multitude of environmental signals to make important cell-fate decisions and fulfill their effector functions. These processes are initiated and regulated by a diverse array of immune receptors and via their dynamic spatiotemporal organization upon ligand binding. Given the widespread relevance of the immune system to health and disease, there have been significant efforts toward understanding the biophysical principles governing immune receptor signaling and activation, as well as the development of biomaterials which exploit these principles for therapeutic immune engineering. Here, how advances in the field of DNA nanotechnology constitute a growing toolbox for further pursuit of these endeavors is discussed. Key cellular players involved in the induction of immunity against pathogens or diseased cells are first summarized. How the ability to design DNA nanostructures with custom shapes, dynamics, and with site-specific incorporation of diverse guests can be leveraged to manipulate the signaling pathways that regulate these processes is then presented. It is followed by highlighting emerging applications of DNA nanotechnology at the crossroads of immune engineering, such as in vitro reconstitution platforms, vaccines, and adjuvant delivery systems. Finally, outstanding questions that remain for further advancing immune-modulatory DNA nanodevices are outlined.

The γ-Protocadherins Regulate the Survival of GABAergic Interneurons during Developmental Cell Death.

Inhibitory interneurons integrate into developing circuits in specific ratios and distributions. In the neocortex, inhibitory network formation occurs concurrently with the apoptotic elimination of a third of GABAergic interneurons. The cell surface molecules that select interneurons to survive or die are unknown. Here, we report that members of the clustered Protocadherins (cPCDHs) control GABAergic interneuron survival during developmentally-regulated cell death. Conditional deletion of the gene cluster encoding the γ-Protocadherins (Pcdhgs) from developing GABAergic neurons in mice of either sex causes a severe loss of inhibitory populations in multiple brain regions and results in neurologic deficits such as seizures. By focusing on the neocortex and the cerebellar cortex, we demonstrate that reductions of inhibitory interneurons result from elevated apoptosis during the critical postnatal period of programmed cell death (PCD). By contrast, cortical interneuron (cIN) populations are not affected by removal of Pcdhgs from pyramidal neurons or glial cells. Interneuron loss correlates with reduced AKT signaling in Pcdhg mutant interneurons, and is rescued by genetic blockade of the pro-apoptotic factor BAX. Together, these findings identify the PCDHGs as pro-survival transmembrane proteins that select inhibitory interneurons for survival and modulate the extent of PCD. We propose that the PCDHGs contribute to the formation of balanced inhibitory networks by controlling the size of GABAergic interneuron populations in the developing brain.SIGNIFICANCE STATEMENT A pivotal step for establishing appropriate excitatory-inhibitory ratios is adjustment of neuronal populations by cell death. In the mouse neocortex, a third of GABAergic interneurons are eliminated by BAX-dependent apoptosis during the first postnatal week. Interneuron cell death is modulated by neural activity and pro-survival pathways but the cell-surface molecules that select interneurons for survival or death are unknown. We demonstrate that members of the cadherin superfamily, the clustered γ-Protocadherins (PCDHGs), regulate the survival of inhibitory interneurons and the balance of cell death. Deletion of the Pcdhgs in mice causes inhibitory interneuron loss in the cortex and cerebellum, and leads to motor deficits and seizures. Our findings provide a molecular basis for controlling inhibitory interneuron population size during circuit formation.