Angling for Novel Gene Expression Data with weMERFISH
Summary by Matt Monaghan: Wan, Y., El Kholtei, J., Jenie, I., Colomer-Rosell, M., Liu, J., Acedo, J.N., Du, L.Y., Codina-Tobias, M., Wang, M., Sawh, A., Lin, E., Chuang, T., Mango, S.E., Yu, G., Bintu, B., Schier, A.F. (2024). Whole-embryo Spatial Transcriptomics at Subcellular Resolution from Gastrulation to Organogenesis. bioRxiv. 10.1101/2024.08.27.609868.
Image credit: Matt Monaghan
A long-standing question is how embryogenesis is orchestrated by gene expression and cellular differentiation. A recent preprint from the Schier lab describing a “whole-embryo imaging platform using multiplexed error-robust fluorescent in-situ hybridization (weMERFISH)” aims to explain spatial gene expression in the vertebrate embryo, as previous methods can lack direct spatial information [1]. An ideal dataset that would help us understand development includes cell-by-cell knowledge of what genes are expressed, what gene regulatory elements are accessible, where those cells are in the context of the embryo, and how those cells interact with their neighbors. Although different methods can provide information about aspects of gene expression with varying levels of resolution, it has been challenging to create a comprehensive picture of how these processes work together. Wan et al. developed a strategy to combine data from different assays to create a more comprehensive picture of spatial gene regulation in the zebrafish embryo.
First, to generate a comprehensive spatial map of gene regulation in embryos, Wan et al. have developed an in-situ hybridization strategy to visualize gene expression known as weMERFISH, based from MERFISH [2, 3]. In weMERFISH, a complementary “primary” [1] probe binds a specific RNA. Next, the sample is covered in a polyacrylamide gel. The primary probe is firmly attached to the gel with a special modification. Next, a “linker” [1] probe specific to the primary probe can be reversibly attached to recruit fluorophores for imaging. Then, the linker probe can be removed and new linker probes specific to other RNAs can be added. All of this can be done in a single embryo and repeated. This strategy was used to directly assess expression of 495 genes at three zebrafish embryonic stages. However, the zebrafish genome contains over 20,000 genes. To solve this problem, the authors made an informed inference or “imputed” [1] spatial expression data for the remaining genes. The authors draw from two data sets: (1) weMERFISH data providing spatial information for just ~500 genes and (2) single-nuclei RNA-seq (snRNA-seq) data describing expression of all genes without direct spatial information. Imputation is done by grouping cells based on RNA present in both weMERFISH and snRNA-seq data, using weMERFISH data to identify the spatial location of the cells, then assessing expression of remaining genes not measured by weMERFISH at that location.
The authors then investigated how genes are regulated at the DNA level. snATAC-seq was performed in the same embryos used for snRNA-seq. ATAC-seq maps open chromatin to identify candidate regions of DNA involved in regulating gene expression. These open regions were then imputed onto the spatial embryo map, providing a 3D model of genome accessibility.
The data is available in an online atlas, MERFISHEYES. Users can explore maps of spatial gene expression and open chromatin at different stages. MERFISHEYES could become an integral part of developmental biology research by providing insight into gene expression and regulation in zebrafish. This study sheds light on some of the ‘what, when, where and why’ connecting gene expression and embryo development.
1. Wan, Y., et al., Whole-embryo Spatial Transcriptomics at Subcellular Resolution from Gastrulation to Organogenesis. Biorxiv, 2024.
2. Moffitt, J.R., et al., High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proc Natl Acad Sci U S A, 2016. 113(50): p. 14456-14461.
3. Moffitt, J.R. and X. Zhuang, RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH). Methods Enzymol, 2016. 572: p. 1-49.