Temporal effects of BMP4 on mouse embryonic and extraembryonic development

This repository is the accompanying code for our paper on the development of extraembryonic ectoderm and embryonic tissues during mouse gastrulation. There is an MCView shiny app where you can interrogate the data. The code is splitted into jupyter notebooks that can be found in the notebook folder.

Quick links

• Interactive extraembryonic ectoderm and embryonic atlases

• Metacells paper: Ben-Kiki et al. 2022 Genome Biology

• Metacells python package

• [MCView] (https://github.com/tanaylab/MCView)

• Raw FASTQ files are available under GEO accessions GSE267870

Running the notebooks

Prior to any analysis, after cloning the repository, please download first the necessary data by running (in the root directory of the cloned repository):

R -e "source('scripts/download_data.R'); download_full_data()"

This will download all the necessary data including processed metacell objects necessary for generating the figures. The subfolder data/umi.tables contains all the UMI matrices of the scRNA-seq data used in the paper.

Necessary R packages

The initialization script (scripts/init.R) loads automatically the necessary R packages to run the notebooks. The analysis was done using R 4.0.5 and the following packages:

• devtools_2.4.2
• usethis_2.0.1
• here_1.0.1
• slanter_0.2-0
• DoubletFinder_2.0.3
• SeuratObject_4.0.2
• Seurat_4.0.3
• forcats_0.5.1
• stringr_1.4.0
• dplyr_1.0.9
• purrr_0.3.4
• readr_2.1.0
• tidyr_1.2.0
• tibble_3.1.3
• ggplot2_3.3.5
• tidyverse_1.3.1
• umap_0.2.7.0
• tgutil_0.1.13
• tgstat_2.3.17
• metacell_0.3.7
• Matrix_1.3-4
• data.table_1.14.2
• qvalue_2.22.0
• princurve_2.1.6
• RColorBrewer_1.1-2
• tglkmeans_0.3.4
• zoo_1.8-9
• ggrepel_0.9.1

Notebook order

For every figure there is a corresponding notebook that generates the plots shown in the figure. In addition, the notebooks and scripts below were run prior to the final data analysis steps. It is not necessary to run those for reproducing specific figures. If you want to rerun analysis steps prior to the figure generation, you should follow the order of the notebooks below.

Analysis of wildtype extra-embryonic ectoderm and embryonic manifolds

For reproducing the analysis of the wildtype ExE and embryonic manifold you should run the following notebooks in that order.

1. import_mars
2. embexe_find_bad_genes
3. embexe metacell construction
4. embryo_temporal_ordering
5. embexe_interpolate_time
6. mc2d_projection_embexe
7. split_embexe_into_emb_and_exe
8. emb.estimation_of_proliferation_rates
9. emb_generate_network
10. mc2d_projection_emb
11. mc2d_projection_exe

Analysis of Bmp4 KO embryos

1. import_embexe_bmp4_og2_plates.ipynb - notebook for importing MARS-seq plates from WT, OG2 and Bmp4 experiments
2. metacell2_embexe_bmp4_og2.ipynb - notebook for creating a joint metacell object
3. embexe_bmp4_generate_cgraph_and_cell_type_time_annotation.ipynb - notebook for transferring cell type annotation from wildtype atlas to Bmp4 KO cells
4. Find_lateral_genes_Bmp4_vs_WT.ipynb - differential expression analysis per cell type between KO, control and WT cells. Used to find and filter genes that are also differentially expressed in control embryos

Analysis of EXE-specific Elf5 KO embryos

10. import_elf5
11. elf5_processing_summary

Analysis of ex utero cultured embryos

12. import_10x_exutero - initial import of scRNA-seq data from ex utero cultured embryos
13. exutero_doublet_removal - doublet removal using DoubletFinder
14. exutero_f_find_bad_genes - remove genes from selected genes for metacell construction that are associated with
15. exutero_f_generate_metacell - generate metacell1 object
16. mc2d_projection_exutero_f - 2d projection of metacell1 object
17. wt_atlas_projection_of_exutero_embryos - atlas projection of ex utero data on WT atlas
18. atlas_self_projection_of_wt_cells

Contact

For help, please contact [email protected] or [email protected] .