Each year, 9 months of dreams and anticipation shared by millions of parents-to-be turn to despair and fright when learning their child is born with a birth defect; an often-devastating event affecting one out of 20 children born worldwide. The formation of our organs, limbs, and face are the result of carefully choreographed movement and behavior by millions of cells, much like dancers in a troupe. If even a few cells don't get to the right position and do their job correctly, the end result is a birth defect. Yet, how each individual cell knows what to do at precisely the right time and place has largely been a mystery.

In a new study published in the scientific journal Nature, a team of researchers reveal for the first time the full spectrum of cells that come together to make a heart at the earliest stages of embryo formation. They also uncovered how the cells are controlled, and how a mutation in just one gene can have catastrophic consequences by affecting a tiny group of cells that make up the organ.

Congenital heart defects are the most common and most lethal human birth defect. Thanks to the advent of a powerful new technology known as single-cell RNA sequencing, the researchers were finally able to discern the role of tens of thousands of individual cells during the formation of the heart, which is essential to determine how genetic mutations cause disease.

"With genome sequencing, we can now more easily find genetic variants that we think are contributing to a disease," said the Senior Investigator. "The big challenge is figuring out the specific cell type in which this variant is functioning and how those cells are impacted. This has been particularly difficult for birth defects, given that genetic variants affect only a small subset of the cells in the organ. With single-cell technologies, we can finally begin to unravel the mechanisms behind defects for which we know the genetic cause."

The catalog the team compiled contains all the genes that are active during different stages of heart development and identifies the cells in which they can be found. It represents the first step in making the connection between a genetic variant and a specific cell type.

"This can tell us, among many other things, which subset of cells are performing critical functions in specific regions of the heart and which are contributing to the underlying cause of a disease associated with genetic mutations," explained the first author of the study.

To complete the repository, the researchers studied nearly 40,000 individual cardiac cells from a mouse model of heart development. The technology that made this study possible is single-cell RNA sequencing. This sophisticated method, which has only been commercially available for the past 3 years in its current form, enabled the scientists to capture data about thousands of individual cells at once.

Once they identified the numerous types of cells involved in heart development, the team wanted to learn how these diverse cell types are generated. To do so, they teamed up with computational biologists who specialize in using data from single-cell RNA sequencing to uncover the molecular drivers of different cell types.

The computational analysis predicted the genes involved in generating specific cell types in the heart, which shed light on those cells' function. The analysis also pointed to one major player, a gene called Hand2 that can control the activity of thousands of other genes.

By applying single-cell RNA sequencing, the teams were able to get a much more detailed and complete picture of how the loss of Hand2 causes different cell populations to become dysregulated.

Mice lacking the gene Hand2 fail to form the right ventricle chamber, which pumps blood to the lungs. Surprisingly, the new prediction made by the researchers suggested that Hand2 is not required for cells being instructed to become right ventricular cells, but that it is critical in forming the cells of the outflow tract, the structure where major outgoing blood vessels of the heart arise.

"This didn't make sense based on previous findings," said the author. "However, we found that, in fact, Hand2 has very distinct functions for different cell types."

The computational prediction turned out to be correct. The team discovered that hearts without the Hand2 gene never made cells of the outflow tract, but did make right ventricular cells. In the choreography of the heart, it is not enough for a cell to be made, it must also get to the right place relative to the other "dancers." Without Hand2, right ventricle cells were created but stuck at their origin, failing to move into the developing heart.

"Our collaborative findings made us change the way we think about heart formation, and showed how disruption of cell fate, migration, or survival of a few cells can cause a heart defect," the author added.

Significantly, the new catalog of cardiac cells can now serve scientists and physicians interested in various aspects of heart development. With knowledge of the types of cells involved in normal and abnormal formation of the heart, the scientific community can begin to design strategies to correct genetic variants that cause congenital heart disease.

These findings could also guide therapeutic approaches to help both newborns and the growing adult population with congenital heart disease.

https://www.nature.com/articles/s41586-019-1414-x