Albrecht, N. E. | bioRxiv Science

bioRxiv2021

Rapid 3D-STORM imaging of diverse molecular targets in tissue

The precise organization of fine scale molecular architecture is critical for the nervous system and other biological functions and would benefit from nanoscopic imaging methods with improved accessibility, throughput, and native tissue compatibility. Here, we report RAIN-STORM, a rapid and scalable imaging approach that enables three-dimensional nanoscale target visualization for multiple subcellular and intracellular targets within tissue at depth. RAIN-STORM utilizes conventional tissue samples, readily available reagents in optimized formulas, requires no specialized sample handling, and is suitable for commercial instrumentation. To illustrate RAIN-STORMs ability for quantitative high-resolution nanoscopic tissue imaging, we utilized the well-organized but structurally complex retina. We show that RAIN-STORM is rapid and versatile, enabling 3D nanoscopic imaging of over 20 distinct targets to reveal known and novel nanoscale features of synapses, neurons, glia, and vascular. Further, imaging parameters are compatible with a wide range of tissue sources and molecular targets across a spectrum of biological structures. Finally, we show that this method can be applied to clinically derived samples and reveal the nanoscale distribution of molecular targets within human samples. RAIN-STORM thus enables rapid 3D imaging for a range of molecules, paving the way for high throughput studies of nanoscopic molecular features in intact tissue from diverse sources.

neuroscience↗

bioRxiv2019

Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

Neuron function relies on and instructs the development and precise organization of neurovascular units that in turn support circuit activity. However, our understanding of the molecular cues that regulate this relationship remains sparse. Using a high-throughput screening pipeline, we recently identified several new regulators of vascular patterning. Among these was the potassium channel tetramerization domain-containing protein 7 (KCTD7). Mutations in KCTD7 are associated with progressive myoclonic epilepsy, but how KCTD7 regulates neural development and function remains poorly understood. To begin to identify such mechanisms, we focus on mouse retina, a tractable part of the central nervous system that contains precisely ordered neuron subtypes supported by a trilaminar intravascular network. We find that deletion of Kctd7 results in defective patterning of the adult retina vascular network, resulting in increased branching, vessel length, and lacunarity. These alterations reflect early and specific defects in vessel development, as emergence of the superficial and deep vascular layers were delayed. These defects are likely due to a role for Kctd7 in inner retina neurons. Kctd7 it is absent from vessels but present in neurons in the inner retina, and its deletion resulted in a corresponding increase in the number of bipolar cells in development and increased vessel branching in adults. These alterations were accompanied by retinal function deficits. Together, these data suggest that neuronal Kctd7 drives growth and patterning of the vasculature and suggest that neurovascular interactions may participate in the pathogenesis of KCTD7-related human diseases.\n\nAlevy et al. Highlights O_LIKctd7 is required for normal retinal vascular organization and retinal function in adults.\nC_LIO_LIDeletion of Kctd7 disrupts the emergence of the superficial and deep vessel layers.\nC_LIO_LIKctd7 may impact vascular patterning through influencing neurons as it is expressed in and regulates bipolar cells.\nC_LIO_LIKctd7 driven neurovascular interactions may participate in the pathogenesis of KCTD7-related human diseases.\nC_LI\n\nLay SummaryNeuron function requires an organized vasculature to maintain brain health and prevent disease, but many neurovasculature regulatory genes remain unknown. Alevy et al. identify the progressive myoclonic epilepsy-associated gene Kctd7 as a key regulator of vascular development and retinal function. They further show that Kctd7 regulation of vessel patterning likely occurs downstream of its role in regulating the development or activity of specific neuron types. These data suggest that KCTD7-regulated neurovascular interactions may participate in the pathogenesis of associated human diseases.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC=\"FIGDIR/small/647008v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (37K):\norg.highwire.dtl.DTLVardef@176b8eorg.highwire.dtl.DTLVardef@1967120org.highwire.dtl.DTLVardef@1dcf4e5org.highwire.dtl.DTLVardef@151fce2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗