Researchers at Harvard Medical School and the Institute of Science and Technology (IST) Austria have discovered a key control mechanism that cells use to self-organize during early embryonic development. Through studies of spinal cord formation in zebrafish embryos, the team found that different cell types expressed unique combinations of adhesion molecules in order to self-sort during morphogenesis. Such “adhesion codes” determined which cells preferred to stay connected, and how strongly they did so, even as widespread cellular rearrangements occurred in the developing embryo. These adhesion codes were found to regulated by morphogens, master signaling molecules long known to govern cell fate and pattern formation in development.

The combined study results suggest that the interplay of morphogens and adhesion properties allowed cells to organize with the precision and consistency required to build a complete organism. The new findings shed light on a process that is fundamental to multicellular life, and could open up new avenues for improved tissue and organ engineering strategies.

Live-cell imaging shows the dynamic environment and extent of cell movement that occurs as the nascent spinal cord is formed during early development. [Tony Tsai/Sean Megason/Harvard Medical School]

“My lab’s goal is to understand the basic design principles of biological form,” said Sean Megason, PhD, professor of systems biology in the Blavatnik Institute at HMS, “Our findings represent a new way of approaching the question of morphogenesis, which is one of the oldest and most important in embryology. We see this as the tip of the iceberg for such efforts.” Megason is co-corresponding author of the team’s study, which is published in Science.

Insights into how cells self-organize in early development could also aid efforts to engineer tissues and organs for clinical uses such as transplantation, the authors said. “Constructing artificial tissues for research or medical applications is a critically important goal, but currently one of the biggest problems is inconsistency,” added study first author Tony Tsai, PhD, research fellow in systems biology in the Blavatnik Institute. “There is a clear lesson to learn from understanding and reverse engineering how cells in a developing embryo are able to build the components of an organism in such a robust and reproducible way.”

Megason, Tsai and colleagues report on their findings in a paper titled “An adhesion code ensures robust pattern formation during tissue morphogenesis.”

Under a microscope, the first few hours of every multicellular organism’s life seem incongruously chaotic. After fertilization, the single-celled egg divides again and again, quickly becoming a visually tumultuous mosh pit of cells jockeying for position inside the rapidly growing embryo.

Yet, amid this apparent pandemonium, cells begin to self-organize. Soon, spatial patterns emerge, serving as the foundation for the construction of tissues, organs and elaborate anatomical structures from brains to toes and everything in between. As the authors noted, “Animal development entails the organization of specific cell types in space and time, and spatial patterns must form in a robust manner.” This imperative to organize is just as true in the zebrafish, which is a common model for developmental studies. “In the zebrafish spinal cord, neural progenitors form stereotypic patterns despite noisy morphogen signaling and large-scale cellular rearrangements during morphogenesis and growth,” the authors stated.

While scientists have intensively studied this organizational process—morphogenesis—it remains in many ways enigmatic. Spearheaded by Tsai, and in collaboration with Carl-Philipp Heisenberg, PhD, and colleagues at IST Austria, the research team first looked at one of the most well established frameworks for morphogenesis, the French flag model. In this model, morphogens are released from localized sources in the embryo, exposing nearby cells to higher levels of the signaling molecule than cells farther away. The amount of morphogen a cell is exposed to activates different cellular programs, particularly those that determine cell fate. Concentration gradients of morphogens therefore “paint” patterns onto groups of cells, evocative of the distinct color bands of the French flag.

“Spatial patterns of distinct cell types arise reproducibly in development,” the authors commented. “The classic French flag model posits that a morphogen gradient forms across a naïve and static field of cells to provide positional information to specify patterned cell fates. The vertebrate spinal cord has been a textbook example of the French flag model.”

This model does have limitations, however. Previous studies from the Megason lab used live-cell imaging and single-cell tracking in whole zebrafish embryos to show that morphogen signals can be noisy and imprecise, particularly at the boundaries of the “flag.” In addition, cells in a developing embryo are constantly dividing and in motion, which can scramble the morphogen signal. This results in an initial mixed patterning of cell types.

Nevertheless cells do self sort into precise patterns, even with a noisy start, the researchers noted. “… the stereotypic stripe patterns still form reproducibly. This makes the zebrafish spinal cord an attractive system to study how robust patterning can be achieved despite imprecision in morphogen signaling and extensive cell-cell neighbor exchange during tissue morphogenesis and growth.”

For their studies, the team set out to understand how this patterning occurs. They focused on a hypothesis proposed over 50 years ago, known as differential adhesion. This model suggests that cells adhere to certain other cell types, self-sorting in a way similar to how oil and vinegar separate over time. But there was little evidence that this plays a role in patterning.

To investigate this in more detail, Megason, Tsai and colleagues developed a method to measure the force by which cells adhere to one another. They placed two individual cells together and then pulled on each cell with precisely controlled suction pressure from two micropipettes. This allowed the researchers to measure the precise amount of force needed to pull the cells apart. By analyzing three cells at once, they could also establish adhesion preferences. “To enable more direct comparison of adhesion preferences, we developed a triplet competition assay,” they explained. The triplet is composed of two cells of the same type and one cell of a different type. “When the triplet is pulled apart, it mimics the challenge faced by the cells in vivo when they are pulled by neighboring cells in different directions.”

The team used this technique to study the patterning of three different types of neural progenitor cells involved in building the nascent spinal cord in zebrafish embryos. The experiments revealed that cells of a similar type strongly and preferentially adhered to one another. To identify the relevant adhesion molecule-encoding genes, the researchers analyzed the gene expression profile of each cell type using single-cell RNA sequencing. They then used CRISPR-Cas9 to block the expression of candidate genes, one at a time. If pattern formation became disrupted, they applied the pulling assay to see how much the molecule contributed to adhesion.

A micropipette assay measures adhesion force between two cells. [Tony Tsai/Sean Megason/Harvard Medical School]


Three genes – N-cadherin, cadherin 11, and protocadherin 19 – emerged as essential for normal patterning. “N-cadherin (cdh2), cadherin 11 (cdh11), and protocadherin 19 (pcdh19) stood out as genes with significant loss-of-function phenotypes,” the scientists wrote. The expression of different combinations and different levels of these genes was responsible for differences in adhesion preference, representing what the team dubbed an adhesion code. This code was unique to each cell type and determined which other cells each cell type stayed connected to during morphogenesis. “Together, quantitative analyses of cdh2, cdh11, and pcdh19 expression revealed an adhesion code specific to each of the three cell types … ” the scientists further commented.

“All three adhesion molecules we looked at are expressed in different amounts in each cell type,” Tsai said. “Cells use this code to preferentially adhere to cells of their own type, which is what allows different cell types to separate during pattern formation. But cells also maintain some level of adhesion with other cell types since they have to collaborate to form tissues. By piecing together these local interaction rules, we can illuminate the global picture.”

Because the adhesion code is cell type specific, the researchers hypothesized that it is likely controlled by the same processes that determine cell fate, namely, morphogen signaling. They looked at how perturbations to one the most well known morphogens, Sonic hedgehog (Shh), affected cell type and corresponding adhesion-molecule gene expression. Their analyses revealed that both cell type and adhesion molecule gene expression were highly correlated, both in level and spatial position. This held true across the entire nascent spinal cord, where patterns of gene expression for cell type and adhesion molecule changed together in response to differences in Shh activity.

The authors concluded, “In the zebrafish spinal cord, we show that robust patterning requires a previously unappreciated interplay between two classic ideas for patterning—morphogen gradients and differential adhesion. The morphogen gradient, though not precise, allows cells with similar adhesion properties to be specified near each other; the differential adhesion mechanism then drives local self-organization of cells to correct any imperfection in the initial pattern and to remain organized in domains throughout tissue morphogenesis.”

“What we found is that this morphogen not only controls cell fate, it controls cell adhesion,” Megason added. “The French flag model gives a rough sketch, and differential adhesion then forms the precise pattern. Combining these different strategies appears to be how cells build patterns in 3D space and time as the embryo is forming.”

The researchers are now investigating further the interplay between morphogen signaling and adhesion in developing embryos. While their reported study looked at three different cell types, there are many other adhesion molecule candidates and morphogens that remain to be analyzed, the authors said. In addition, the details of how morphogens control both cell type and adhesion molecule expression remain unclear. Better understanding these processes could help scientists uncover and reverse engineer the fundamental mechanisms by which a single-celled egg constructs a whole organism, they suggested. This could have profound implications in biotechnology, particularly for efforts to build artificial tissues and organs for transplantation or for testing new drug candidates.

“The issue with tissue engineering right now is that we just don’t know what the underlying science is,” Megason acknowledged. “If you want to build a little bridge over a stream, maybe you could do that without understanding physics. But if you wanted to build a big suspension bridge, you need to know a lot about the underlying physics. Our goal is to figure out what those rules are for the embryo.”

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