Simon et al 2025

This repository contains the data and code associated with image analysis in the manuscript Simon et. al., (2025) Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst.

Repository structure and useage

/ImageJ

ImageJ *.ijm macro scripts for batch running StarDist segmentation on confocal microscopy images of pre-implantation human embryos for later quantification in Cell Profiler

For use with StarDist 2D plug-in (Schmidt et. al., 2018).

Modified pipeline from https://github.com/todd-fallesen/Niakan_Lab_KLF17 (Lea et. al., 2021)

/Cell_Profiler

Pipelines for the segmentation and measurement of cytoplasmic and nuclear fluorescence intensities of ImageJ processed confocal microscopy images of pre-implantation human embryos.

• FGF_ERKi_NANOG_GATA4_GATA3_nuclear_intensity.cppipe - pipeline used for nuclear intensity quantification for FGF and ERKi experiments

• pERK_SOX2_OTX2_cyto_nuclear_intensity.cppipe - pipeline used for cytoplasmic and nuclear intensities for pERK experiments

These scripts were compiled in CellProfiler v4.1.3 (McQuin et. al., 2018).

Modified pipeline from https://github.com/todd-fallesen/Niakan_Lab_KLF17 (Lea et. al., 2021)

/data

Pre-processed and processed data for image analysis in R

/embryo_example - pre-processed data to be used with ../R/processing.R - examples of Cell Profiler data outputs, and manual annotations for ICM cells and corrections for under segmentation.

• *.csv - all processed data containing experiment metadata, raw and Z-corrected fluorescence intensitities for each of the FGF, ERKi and pERK experiments.

For raw imaging data, please see Figshare repository DOI dx.doi.org/10.6084/m9.figshare.28597145

/R

R scripts for data processing, analysis and figure generation

• processing.R - processing image quantification from Cell Profiler data output, combining with manual annotations and corrections, and performing Z-correction on fluorescence intensities

  • ebcor.R - function for Empirical Bayes correction of Z-axis associated fluorescence decay. Modified from https://github.com/nestorsaiz/saiz-et-al_2016 (Saiz et. al., 2016)

• *_analysis.R - analysis and figures of FGF or ERK treatments, or pERK experiments

  • *_data.R - load processed experimental data

  • *_hclust.R - hierarchical clustering of nuclear fluorescence intensities to assign ICM lineage identities. Modified from https://github.com/nestorsaiz/saiz-et-al_2020 (Saiz et. al.

These scripts were compiled in R Studio version 2023.12.1+402, and R version 4.3.3

/RNAseq

Processed data and code for bulk and single cell RNAseq analysis. See https://github.com/brickmanlab/simon-et-al-2024 for full code for processing and analysis of single cell RNAseq data, including integration with reference datasets and ML model.