Single-cell, whole-embryo phenotyping of mammalian developmental disorders

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be ‘decomposable’ through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.


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Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
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Sample size
We did not perform prior explicit calculations for sample size.Sample size was determined by availability of embryos from the crossings.We sampled either 50/50 female and male embryos (C57BL/6, BALB/C, FVB) or male only (G4) from the respective genotypes.We included 4 replicates per genotype of mutants and corresponding wildtypes at the embryonic stage E13.5 in the study.
Data exclusions We excluded the embryos 104 and 41 after the quality control step of the analysis from downstream analysis for reasons of low cell number and/or quality of the sample.Sample Nr. 70 was lost in transport prior to the start of the experiment.

Replication
For the sci-RNA-seq3 experiment we isolated nuclei from 103 embryos staged E 13.5, 4 replicates each genotype including 22 mutant backgrounds and the corresponding 4 WT backgrounds.The attempts at replication were successful.
Randomization To minimize batch effects, the nuclei extraction from embryos was randomized.For the first round of indexing, nuclei from each embryo were deposited in seperate wells respectively, such that the first index could be linked to the individual embryos isolated from.After the first round of indexing, all samples were pooled and distributed randomly across four plates for the second indexing round.

Blinding
Investigators were blinded to group allocation during data collection and analysis: Embryo collection, nuclei isolation, library preparation and sci-RNA-seq3 analysis all were performed by different researchers, respectively.

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March 2021 Ecological, evolutionary & environmental sciences study design All studies must disclose on these points even when the disclosure is negative.

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We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response., past and current diagnosis and treatment categories).If you filled out the behavioural & social sciences study design questions and have nothing to add here, write "See above."RecruitmentDescribehow participants were recruited.Outline any potential self-selection bias or other biases that may be present and how these are likely to impact results. information