ABOVE: An artist’s rendering of human embryonic stem cells © ISTOCK.COM, ANUSORN NAKDEE

The primitive streak—a structure that emerges in mammalian and avian embryos to help assign spatial information to cells during early development—has long been regarded as essential for the genesis of an organism’s body plan and individuality, and was thus used as a landmark in ethical guidelines for stem cell research. But a new literature review pulls together evidence from stem cell research, evolutionary history papers, and developmental biology studies to make the argument that the primitive streak may not be so necessary after all. In doing so, the review—which was published today (December 2) in Science—challenges century-old assumptions that scientists have been making regarding how an embryo changes from a collection of identical cells into an organism with a distinct, asymmetric form.

The new review suggests that the streak is “unnecessary” and “dispensable,” University of Michigan developmental biologist and biomedical engineer Jianping Fu, who didn’t work on the review but performed research that the review cited, tells The Scientist. He adds that he found the review “refreshing” for its ability to start new discussions about embryonic development and challenge dogmatic thinking that has dictated the conversation for years.

The review’s conclusion that the streak isn’t essential, if further supported, could provide the impetus to change the way stem cell and embryonic research experiments are regulated. Back in 1990 the United Kingdom imposed the first version of what’s called the “14-day rule,” a regulation stating that any human embryos cultured by researchers must be terminated after the primitive streak forms (at two weeks post fertilization in human embryos) that’s since become an influential international ethical guideline. While the International Society for Stem Cell Research (ISSCR) relaxed the rule in May 2021, allowing experiments to continue longer on a case-by-case basis, a two-week timeframe is still the widely-accepted norm.

If that were to change as a result of the paper, it could result in a “profound” change in biomedical research, Fu says. Specifically, he says it could justify research into developmental diseases or other conditions that researchers couldn’t explore in depth previously because of the 14-day limit. “This will open the door for continuous progress to use human stem cells to generate human embryo models,” he adds.

A primitive marker of a being

The primitive streak forms at the beginning of a developmental stage called gastrulation—a catch-all term for the part of early development when a ball of epithelial cells called a blastula transitions into a three-dimensional structure called a gastrula, which contains multiple layers of different cell types. Gastrulation is also when symmetry across the dorsal-ventral and anterior-posterior axes begins to emerge. Cells that will eventually end up as part of an animal’s head, back, and spine—assuming the animal in question has them—differentiate during this process.

At the beginning of gastrulation, a furrowlike structure appears in the embryos of birds and nearly all mammals—monotremes, which include platypuses and echidnas, are likely excluded. This is the primitive streak. In humans, the primitive streak emerges in the blastula’s outer layer—the epiblast—14 days after fertilization, as a crease that establishes the embryo’s anterior-posterior axis.

For decades, researchers have considered the primitive streak to be a crucial player in allocating and transporting epithelial pluripotent stem cells into one of the three layers of specialized cells that forms during gastrulation and ultimately give rise to all the different kinds of tissue in the adult organism, the new paper explains. However, the review makes the argument that the primitive streak is a byproduct of the gastrulation process, not the shepherd of epithelial cell allocation and.

The papers cited in the review “really called into question ‘is this structure something that is an absolute requirement for giving cells their identity?’” review coauthor and University of Virginia developmental biologist Ann Sutherland tells The Scientist. “Or is it just a convenient way to get the cells from one place to another, and that final destination is where they’re going to differentiate and create new tissue shapes?”

That’s the crux of the review’s controversial prediction: that the primitive streak is a useful evolutionary trick, but not a necessity for embryonic development.

The review’s authors cite the fact that many branches of the animal kingdom don’t seem to develop a primitive streak—and the fact that mammals and birds evolved to have primitive streaks independently of one another—as evidence of the structure’s arbitrariness.

Most people in the field recognize that the 14-day rule was somewhat an arbitrary cutoff.

—Aryeh Warmflash, Rice University

Rice University cell biologist Aryeh Warmflash, who didn’t contribute to the review but whose work is heavily cited as evidence for the authors’ hypothesis, tells The Scientist that arguing about whether the primitive streak is necessary for gastrulation may simply be a matter of semantics, because the primitive streak only pertains to some of the many aspects of the overall gastrulation process. For the rest, he says, the primitive streak was already known to be irrelevant.

It is true that many animals that go through gastrulation do so without a primitive streak, Warmflash points out, and there are also many aspects of gastrulation that occur independently of the primitive streak among the animals that do develop them. So, “if you’re talking about differentiation into dermal layers, it’s clear you don’t need any sort of organized structure” such as the primitive streak, Warmflash says, adding that the primitive streak does seem to be necessary for establishing “spatial structure in the embryo.”

But that doesn’t necessarily refute its importance in species where it does occur, he says. “Whether those processes could be decoupled in embryos that have a primitive streak is not totally clear.”

The review also points to experimental evidence—including studies from Warmflash’s lab and work done by the review’s coauthors—from in vitro pluripotent models using human and mouse stem cells in which cell differentiation occurs and a rudimentary body plan often emerges without a primitive streak appearing.

Warmflash developed a 2D stem cell model of embryonic development that resulted in organization and structures that resembled a real gastrula. The trick to getting cells to differentiate into tissue resembling the “very orderly” embryo rather than a nondescript “mass” was to physically restrict the ways in which cells could grow.

Sutherland says that while these stem cell models have been inconsistent and that questions remain, her interpretation of their evidence to date is that the primitive streak isn’t a structure that forms in order to drive gastrulation. Rather, it is something that appears based on physical and mechanical constraints imposed on the embryo.

See “Researchers Generate Model of Human Embryo from Human Stem Cells

She explains that primitive streaks appear different from one species to the next, and suggests that this variation may stem from differences in the embryo—factors like yolk size and tautness of the epiblast—and in the surrounding environment. Extending the argument further, the review suggests that the primitive streak appears or doesn’t appear in certain species due to those factors, and is therefore not a developmental necessity.

“We suggest that all embryos acquire a midline very early on that will organize the emerging cell types and that the primitive streak is a mechanical response of the cells along this line created by the interactions of the embryo with the extraembryonic tissues,” review coauthor Alfonso Martinez Arias, a stem cell researcher at the University of Cambridge and Pompeu Fabra University, tells The Scientist via email. “The [stem cell] based models lack extraembryonic tissues and thus lack the primitive streak.”

“What we do here is also redefine gastrulation as the process that generates the coordinate system that will act as a reference for the organization of tissues and organs,” Martinez Arias adds. “From this perspective, it is perhaps not surprising that . . . there is more than one way to solve this problem.”

Is this structure something that is an absolute requirement for giving cells their identity? Or is it just a convenient way to get the cells from one place to another?

—Ann Sutherland, University of Virginia 

Somewhat ironically, Warmflash remains unconvinced by the analysis, telling The Scientist that it seems unlikely that a theoretical human embryo that fails to create a primitive streak would develop into a person.  “From an organismal level, whether you could make a human being without a primitive streak—I don’t think there’s any real evidence of that.”

Warmflash further questions the use of stem cell models as evidence, noting that the best in vitro stem cell models in use today lack the same spatial geometry and 3D structure of an actual embryo. That makes them poor substitutes for in vivo development, he says, and complicates drawing definitive conclusions on the role of the primitive streak.

If scientists could “demonstrate proper development of germ layers without development of the primitive streak” in a stem cell model, that would serve as definitive proof of the review’s predictions, says Fu.

But that’s easier said than done. “Recapitulating a human gastrula embryo in vitro is a sort of holy grail for many,” review coauthor and Kumamoto University developmental geneticist Guojun Sheng tells The Scientist in an email. He adds that successfully doing so would enable researchers to induce better integrated tissues and organs in their in vitro models. “So whether our prediction is correct or not will become evident if stem cell biologists can (or cannot) achieve [that].”

Where to draw the line

It’s not clear what impact the review’s conclusions may have on stem cell regulation, but experts tell The Scientist that the review helps point out that the 14-day rule was, in some ways, always flawed. Every researcher who spoke to The Scientist about the new review paper agrees that the ISSCR’s updated guidelines are a step in the right direction, and that the original usage of the primitive streak as the basis for the 14-day rule was capricious.

“Most people in the field recognize that the 14-day rule was somewhat an arbitrary cutoff,” Warmflash says. “At the time it was established, scientists didn’t worry that much about it. In terms of research, we were nowhere close to being able to culture embryos past 14 days or to being able to recapitulate these stem cells.” Regulators pushed forward with it because the emergence of the primitive streak was thought to represent the emergence of human individuality, after which it becomes unethical to continue experimentation.

However, the review’s prediction that the primitive streak isn’t necessary makes it an “unreliable ethical red line,” Sutherland says.

While it refutes the primitive streak’s importance in development and therefore its utility as a hard stopping point for human embryo or stem cell research, the review provides no clear answer for what could replace the 14-day rule.

Speaking speculatively, Sutherland suggested the emergence of the heart or brain could serve as a new landmark, and Warmflash suggested that the emergence of the ability to feel pain or of coordinated neural activity resembling consciousness could serve as guidelines. But both say that the answer needs to come from bigger conversations among more diverse communities, including researchers, bioethicists, regulatory agencies, and an engaged public.

“I think, at this point, we really have to think carefully about which ones of those are the best ones to use,” Sutherland says. “I myself don’t feel qualified to make a strong decision or a strong recommendation, because there are a lot of things to think about.”