Single-cell transcriptome analysis during cardiogenesis reveals basis for organ level developmental anomalies

Abstract

Organogenesis involves integration of myriad cell types with reciprocal interactions, each progressing through successive stages of lineage specification and differentiation. Establishment of unique gene networks within each cell dictates fate determination, and mutations of transcription factors that drive such networks can result in birth defects. Congenital heart defects are the most common malformations and are caused by disruption of discrete subsets of progenitors1–3, however, determining the transcriptional changes in individual cells that lead to organ-level defects in the heart, or other organs, has not been tractable. Here, we employed single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular sub-populations can have catastrophic consequences. A network-based computational method for single-cell RNA-sequencing that predicts lineage specifying transcription factors4,5 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite failure of right ventricular formation in Hand2-null mice6. Temporal single-cell transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium differentiated but failed to migrate into the anterior pole of the developing heart. Dysregulation of retinoic acid signaling, responsible for anterior-posterior patterning7, was associated with posteriorization of anterior cardiac progenitors in Hand2-null mutant hearts and ectopic atrial gene expression in outflow tract and right ventricle precursors. This work reveals transcriptional determinants in individual cells that specify cardiac progenitor cell fate and differentiation and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework to investigate congenital heart defects.

Single‐cell transcriptomic landscape of cardiac neural crest cell derivatives during development

“…Notably, the mesenchymal cluster c5, which comprised cells mainly from the early stages (Fig 4B), was inferred by the algorithm to represent the earliest state of the CNCC‐derived mesenchymal cells after migrating into the heart. Consistent with that Crabp1 is the top marker of CNCC‐derived mesenchymal cells at E9.25 (Soysa et al , 2019), c5 showed high expression of Crabp1 and Crabp2 (Appendix Fig S9A and B). As expected, starting from c5, the trajectory was split into two major branches: mesenchymal cell and VSMC developmental branches (Fig 7).…”

Cardioids reveal self-organizing principles of human cardiogenesis

“…In NKX2-5 KO lines, we did not detect any cavity formation defects at the cardiac mesoderm stage and they eventually formed TNNT2 + cardioids (Figures S3A-S3C). HAND1 expression in NKX2-5 KO cardiac mesoderm remained unaffected and this is in line with the delayed onset of NKX2-5 relative to HAND1 expression in cardioids (Figures 1E and S3C) as well as in human and mouse FHF cardiac mesoderm (de Soysa et al, 2019;Tyser et al, 2021). However, HAND1 expression appeared reduced at the CM stage in NKX2-5 KO cardioids (Figure S3C), which is in agreement with findings in mouse and human CMs where NKX2-5 is acting upstream of HAND1 (Anderson et al, 2018;Tanaka et al, 1999).…”

Single-cell transcriptomic landscape of cardiac neural crest cell derivatives during embryonic and neonatal development

“…This subpopulation showed high expression of the Crabp1 gene, encoding cellular retinoic acid binding protein 1 (Figure 6G), which has been reported as the top marker of CNCC-derived mesenchymal cells at E9.25. 42 It also showed high expression of the Crabp2 gene, encoding cellular retinoic acid binding protein 2 (Figure 6H). The expression of Crabp1 and Crabp2 , two important regulators of retinoic acid signaling, decreased during development.…”