Skip to Main Content
It looks like you're using Internet Explorer 11 or older. This website works best with modern browsers such as the latest versions of Chrome, Firefox, Safari, and Edge. If you continue with this browser, you may see unexpected results.

The Levy Library Blog

Article in the Spotlight: April 2022

by Angelyn Thornton on 2022-04-26T08:00:00-04:00 | Comments

 

Each month Levy Library showcases the achievements of Mount Sinai faculty and researchers by highlighting an article and its altmetrics. Altmetrics are alternative measures of impact that capture non-traditional data like abstract views, article downloads, and social media activity. Our altmetrics data is provided by the PlumX platform. 

This month we highlight Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesisThis article was written in part by Evan Bardot.

 

 

ABSTRACT

Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8–12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain–hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal–ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

 

Single-cell spatial transcriptomics map of mouse organogenesis using seqFISH.
a, Illustration of 8–12 ss mouse embryo. Dotted lines indicate the estimated position of the sagittal tissue section shown in b; D, dorsal; V, ventral; R, right; L, left; A, anterior; P, posterior. b, Tile scan of a 20-µm sagittal section of three independently sampled 8–12 ss embryos stained with nuclear dye DAPI (white). Red boxes indicate the selected field of view (FOV) imaged using seqFISH. c, Illustration of the experimental overview for spatial transcriptomics using seqFISH for 351 selected genes in 16 sequential rounds of hybridization and 12 non-barcoded sequential smFISH hybridization rounds for 36 genes. For each targeted gene, 17–48 unique probes were used to capture the mRNA; UMAP, uniform manifold approximation and projection. d, Cell segmentation strategy using a combination of E-cadherin (E-cad), N-cadherin (N-cad), pan-cadherin (Pan-cad) and β-catenin antibody (AB; green) staining detected by an oligo-conjugated anti-mouse IgG secondary antibody (orange) that gets recognized by a tertiary probe sequence. The acrydite group (blue star) of the tertiary probe (blue) gets crosslinked into a hydrogel scaffold and stays in place even after protein removal during tissue clearing. The cell segmentation labeling can be read by a fluorophore-conjugated readout probe (red); AB1, antibody 1; AB2, antibody 2. e, Cell segmentation staining of a 10-µm thick transverse section of an E8.5 mouse embryo using the strategy introduced in d. Cell segmentation signal was used to generate a cell segmentation mask using Ilastik (right). This was repeated independently for all N = 3 embryos with similar results. f, Representative visualization of normalized log expression counts of 12 selected genes measured by seqFISH to validate performance. This experiment was repeated independently for all N = 3 embryos with similar results. g, Highly resolved ‘digital in situ of the cardiomyocyte marker titin (Ttn), Tbx5Cdh5 and Dlk1, colored in red, cyan, green and orange, respectively. Dots represent individually detected mRNA spots, and the box represents an area that was magnified for better visualization. This experiment was repeated independently for all N = 3 embryos with similar results.

 

View this article's PlumX profile here


 Add a Comment

0 Comments.

  Subscribe



Enter your e-mail address to receive notifications of new posts by e-mail.


  Archive



  Follow Us



  Facebook
  Twitter
  Instagram
  Return to Blog
This post is closed for further discussion.

title
Loading...