Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a user-friendly system that can address imaging applications with varied requirements in terms of resolution (mesoscopic to sub-micrometer), sample geometry (size, shape, and number), and compatibility with tissue-clearing protocols and sample holders of various refractive indices. We present a 'hybrid' system that combines a novel non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet architecture for versatile multi-scale volumetric imaging. Main TextRecent advances in tissue-clearing protocols greatly reduce optical scattering, aberrations, and background uorescence, enabling deep-tissue imaging with high resolution and contrast. These approaches have yielded new insights in many elds, including neuroscience, developmental biology, and anatomic pathology [1][2][3][4][5][6][7][8][9][10][11]. Light-sheet microscopy has emerged as a preferred means for highresolution volumetric imaging of cleared tissues due to its unrivaled speed and low photobleaching [12,13]. Many variants of light-sheet microscopes have been developed in recent years by academic researchers and commercial entities to tackle a diverse range of imaging applications (Error! Reference source not found. and Error! Reference source not found.) [14][15][16][17][18]. Whereas individual light-sheet systems are well-suited for a subset of cleared-tissue applications, trade-offs are inevitable. In particular, no current light-sheet microscope can satisfy all of the following requirements: (1) user-friendly mounting
Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently genomically record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-κB, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.
The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.