Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors

Abstract

The scientific community has learned a great deal from imaging small and naturally transparent organisms such as nematodes and zebrafish. The consequences of genetic mutations on their organ development and survival can be visualized easily and with high-throughput at the organism-wide scale. In contrast, three-dimensional information is less accessible in mammalian subjects because the heterogeneity of light-scattering tissue elements renders their organs opaque. Likewise, genetically labeling desired circuits across mammalian bodies is prohibitively slow and costly via the transgenic route. Emerging breakthroughs in viral vector engineering, genome editing tools, and tissue clearing can render larger opaque organisms genetically tractable and transparent for whole-organ cell phenotyping, tract tracing and imaging at depth.

Figure 1

Concept for an in vivo selection technology for panning large-scale libraries to identify compounds or biologicals with optimized physiological properties. Whole-body tissue clearing can then facilitate biodistribution mapping. For example, to engineer viral vectors for more effective transgene delivery, one strategy involves exposing live cells or whole-organisms to AAV capsid libraries and then identifying positive hits via a cell or tissue type-dependent recovery strategy (A). Whole-body clearing by Perfusion-Assisted Agent Release in Situ (PARS, [32,41]) speeds up the multi-organ assessment of vector variants expression profiles. Internal organs before and after clearing (B). Individual PARS-cleared organs (C) before (top) or after (bottom) equilibration in RIMS, a Refractive Index Matching Solution [32,41], as imaging media. Black pointers correspond to the adrenal gland on the kidney, and to the ovaries on the fallopian tubes. Each square represents 0.5 cm². The qualitative assessment of cell-type transduction can be conducted by packaging fluorescent reporters in individual capsid variants and then simultaneously clearing all organs in situ (D). As proof-of-principle, a novel capsid variant (AAV-PHP.B, bottom), selected for enhanced brain transduction, was rapidly evolved from AAV9 (top). Comparisons of PARS-cleared organs demonstrate that AAV-PHP.B and AAV9 have similar cellular tropisms outside of the brain. Arrows (→) indicate neuronal morphology, and asterisks (*) designate pancreatic islets. Differences in brain transduction are depicted in the images of mouse brain sagittal sections. Figures 2A and 2D adapted from [53], and figures 2B–C adapted from [41].

Figure 2

Clearing techniques that enable high-resolution, volumetric imaging of tissue architecture and cellular morphology. Whole-body hydrogel embedding and detergent-based clearing via the PARS-CLARITY method [22,32,37,41] preserves gross tissue structure (A) and fine neuronal processes (B) alike, while the purposeful expansion of these tissue-hydrogel hybrids via water absorption (C) allows the visualization of subcellular detail via either native fluorescence (D), or probes for protein and nucleic acid detection [32]. ePACT permits the clearing and 4-fold expansion of 100 µm thick coronal brain sections with preservation of tissue shape, cellular morphology and native fluorescence. Figures 1A–B adapted from [32], and figures 1C–D adapted from [41].