Biology subjects

Lapan, S. W.

Publications and source records attributed to Lapan, S. W..

5 recordsLinked to original sources

bioRxiv2021

Spatiotemporal patterns of neuronal subtype genesis suggest hierarchical development of retinal diversity

How do neuronal subtypes emerge during development? Recent molecular studies have expanded our knowledge of existing neuronal diversity. However, the genesis of neuronal subtypes remains elusive and previous studies have been limited by a lack of quantitative methods for simultaneous detection of subtype diversity in situ. The bipolar interneurons of the mammalian retina represent a diverse neuronal class, characterized by distinct functions, morphologies, and recently discovered transcriptional profiles. Here, we developed a comprehensive spatiotemporal map of bipolar subtype genesis in the retina. Combining multiplexed detection of 16 RNA markers with timed delivery of EdU and BrdU, we analyzed more than 30,000 single cells in full retinal sections to classify all bipolar subtypes and their birthdates. We found that bipolar subtype birthdates are ordered and follow a centrifugal developmental axis. Spatial analysis revealed a striking oscillatory wave pattern of bipolar subtype birthdates, and lineage analyses suggest clonal restriction on homotypic subtype production. These results inspired a hierarchical model of neuronal subtype genesis in the mammalian retina, with the wave pattern of subtype birthdates arising from early asymmetric cell divisions among founding retinal progenitor cells. Our results provide an outline of the developmental logic that generates diverse neuronal subtypes, and establishes a framework for studying subtype diversification.

developmental biology↗

bioRxiv2019

Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labelled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as the Drosophila midgut. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

molecular biology↗

bioRxiv2018

Highly multiplexed in situ protein imaging with signal amplification by Immuno-SABER

Probing the molecular organization of tissues requires in situ analysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets without in situ enzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.

cell biology↗

bioRxiv2018

SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

Fluorescent in situ hybridization (FISH) reveals the abun-dance and positioning of nucleic acid sequences in fixed sam-ples and can be combined with cell segmentation to produce a powerful single cell gene expression assay. However, it re-mains difficult to label more than a few targets and to visu-alize nucleic acids in environments such as thick tissue sam-ples using conventional FISH technologies. Recently, meth-ods have been developed for multiplexed amplification of FISH signals, yet it remains challenging to achieve high lev-els of simultaneous multiplexing combined with high sam-pling efficiency and simple workflows. Here, we introduce signal amplification by exchange reaction (SABER), which endows oligo-based FISH probes with long, single-stranded DNA concatemers that serve as targets for sensitive fluores-cent detection. We establish that SABER effectively ampli-fies the signal of probes targeting nucleic acids in fixed cells and tissues, can be deployed against at least 17 targets si-multaneously, and detects mRNAs with high efficiency. As a demonstration of the utility of SABER in assays involv-ing genetic manipulations, we apply multiplexed FISH of reporters and cell type markers to the identification of en-hancers with cell type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit to allow rapid and cost effective multiplexed imaging.

genetics↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗