Mammalian Development Disorders Single Cell Whole Embryo Phenotyping

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Summary

In this study, researchers utilized single-cell RNA sequencing on 101 mouse embryos, including 22 mutant and 4 normal types, during a crucial developmental stage (embryonic day 13.5). This innovative method, which examined over 1.6 million nuclei, was aimed at identifying intricate developmental anomalies in mice, a vital step in understanding human developmental disorders. The team focused on a variety of mutations, from complex systemic disorders to specific regulatory region deletions. Their analysis spanned 52 different cell types and developmental pathways, revealing that some mutants affected numerous pathways while others had more localized impacts. Notably, they found commonalities in developmental disruptions across different mutants, hinting at potential decomposable elements of developmental pleiotropy. This comprehensive approach marks a significant advancement in the molecular and cellular profiling of mouse embryos, offering a detailed view of the effects of genetic mutations.

Research Criteria

This article delves into an extensive study involving more than 1.6 million cell nuclei from 101 mouse embryos, using single-cell RNA sequencing to examine both mutant (22 types) and wild-type (4 types) genotypes. The research, conducted on embryos at day 13.5 of development and typically involving four replicates for each strain, sought to pioneer a comprehensive method for evaluating the molecular and cellular characteristics of various mouse genetic models. The range of mutations analyzed varied widely, encompassing everything from well-known complex disorders to specific knockouts of regulatory elements.

Fig.1 Experimental design¹.

Sample Type

Mouse embryos

Result—Single-Nucleus RNA Sequencing of 101 Mouse Embryos

Researchers conducted a comprehensive study on mouse embryos, involving whole-embryo single-nucleus RNA sequencing (snRNA-seq) to evaluate the molecular and cellular characteristics of various genetic models. A total of 103 mouse embryos were examined, including 22 mutants and four wild-type strains, all at embryonic day 13.5. The mutants were selected to represent a range of phenotypic severities, from broad disorders to specific gene knockouts (KOs). The study categorized the mutants into four groups: those affecting multiple organs, those modeling specific human diseases, those with mutations linked to human disease, and those with deletions in cis-regulatory elements. Additionally, the study utilized the mouse organogenesis cell atlas (MOCA) for validation, analyzing over 1.6 million nuclei. Principal component analysis (PCA) revealed two major genetic background groups, and a reference embedding was created for data integration. Ultimately, 13 major developmental trajectories and 59 sub-trajectories were identified, offering insights into cellular development stages.

Fig.2 snRNA-seq of E13.5 complete mouse embryos¹.

Result—Mesenchymal Stalling in Sox9 Regulatory Mutant Altered

The researchers focus on the impact of a specific genetic mutation, the Sox9 regulatory INV, which causes an inversion in a 1-megabase region upstream of the Sox9 gene. This mutation alters the gene's regulatory landscape, impacting the development of various systems, including the skeleton and brain. The mutant shows a pronounced shift in the lochNESS distribution, particularly in mesenchyme, leading to skeletal abnormalities like digit malformation and cleft palate. The inversion also results in a significant reduction in Sox9 expression and a misexpression of the Kcnj2 gene. This misexpression is most notable in the digit anlagen, where Kcnj2 follows a pattern similar to wild-type Sox9. Further investigations reveal changes in cell-type compositions, notably in mesenchymal sub-trajectories, and an increase in Kcnj2 expression across various tissues. The study highlights the complex interplay between genetic mutations and developmental processes, emphasizing the intricate regulatory mechanisms that govern gene expression and their far-reaching implications.

Fig.3 The Sox9 regulatory INV mutant appears to halt and steer mesenchyme differentiation¹.

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Reference

1. Huang, Xingfan et al. "Single-cell, whole-embryo phenotyping of mammalian developmental disorders." Nature, 15 Nov. 2023.