NEW YORK – Researchers have developed a catalog of genes that are expressed during heart development in mice, which they said could be used to better understand how birth defects arise.
Birth defects affect about 5 percent of children, and congenital heart malformations are the most common. Using single-cell RNA sequencing, researchers from the Gladstone Institute of Cardiovascular Disease and elsewhere analyzed nearly 40,000 murine cardiac precursor cells during cardiogenesis. As they reported today in Nature, they found that the transcription factor Hand2 plays a key role in determining cell fate and that its loss in mice contributes to disrupted cardiac development.
"This sequencing technique allowed us to see all the different types of cells present at various stages of heart development and helped us identify which genes are activated and suppressed along the way," senior author Casey Gifford, a staff scientist at Gladstone, said in a statement.
They sequenced more than 36,000 single cells from the cardiogenic region of mouse embryos at three different developmental time points. Broadly, the transcriptomes of the cells could be clustered into seven populations representing different general cell types. Those populations could be further divvied into distinct progenitor cell groups. In all, they generated transcriptomic data representing ventricular, atrial, sinus venosus, atrioventricular canal, and outflow-tract myocardial cells, which the researchers noted represents a catalog of cardiac cell states that arise during heart development.
With this catalog, the researchers were able to delve more deeply into the roles of some of these cardiac progenitor cells.
Using a Boolean-based prediction method, the researchers sought to identify molecular regulators involved in determining the fate of certain cardiac progenitor cells. This computational approach identified Irx4 and Plag1 as specifiers of right ventricle myocardium cells and Hand2, Tead2, and Arid3b as determinants of outflow tract myocardium cell fate.
The researchers noted that the prediction that Hand2 was a lineage specifier for outflow tract myocardium, but not right ventricle myocardium was not expected, since global Hand2 deletion leads the right ventricle to not form properly in mice.
"This didn't make sense based on previous findings," first author Yvanka De Soysa, a graduate student at Gladstone, said in a statement. "However, we found that, in fact, Hand2 has very distinct functions for different cell types."
The researchers examined this discrepancy by comparing the single-cell transcriptomes of wild-type and Hand2-null cardiac progenitor cells at two developmental time points. From this, they found that in Hand2-null cells, anterior heart field, outflow tract, and right ventricle precursor cells were transcriptionally dysregulated early in development, well before any morphological effects would be apparent.
But then they found that though Hand2-null outflow tract-fated cells didn't specify properly, right ventricle-fated cells did. However, after that, the right ventricle cells accumulated in the pharyngeal mesoderm behind the heart, rather than in the cardiac outflow tract.
"Single-cell technologies can inform us about how organs form in ways we couldn't understand before and can provide the underlying cause of disease associated with genetic variations," Gifford said. "We revealed subtle differences in very, very small subsets of cells that actually have catastrophic consequences and could easily have been overlooked in the past. This is the first step toward devising new therapies."