Our results thus show that primitive streak formation is induced by NODAL activation in the posterior hypoblast. The movement of the hypoblast (which is a counter-rotating movement) is a consequence of primitive streak formation – and not a cause of it, as previously thought.

Finally, we investigated whether the anterior hypoblast could inhibit primitive streak formation, as previously thought. By grafting the anterior hypoblast onto the forming primitive streak, we could show that it does not. On the contrary, the grafted hypoblast starts to express NODAL.

In this movie, you can for example see that the full ablation of the hypoblast completely inhibits primitive streak formation, while a partial ablation that leaves a piece of posterior hypoblast on each side leads to the formation of two embryonic axes (forming conjoined twins).

Using HCR-RNA-FISH, we observed that the expression of NODAL, a master activator of primitive streak formation, initiates in the posterior hypoblast and then spreads to the entire posterior blastoderm.

By intercalating a porous filter between the epiblast and the hypoblast, we further showed that the hypoblast requires physical contact with the epiblast to achieve counter-rotating flows. Thus, forces generated in the epiblast propagate by mechanical coupling to the hypoblast, setting it in motion.

By generating a two-color chimera, we observed that the counter-rotating flows of the epiblast and hypoblast are highly synchronized. As a result, the hypoblast and epiblast move very little in relation to each other.

We have adapted live-imaging techniques previously used to characterize tissue flows in the avian epiblast to describe, this time, tissue flows in the hypoblast. This allowed us to discover that, like the epiblast, the hypoblast exhibits counter-rotating flows (also known as Polonaise movements).

In birds and mammals, primitive streak formation is thought to occur when an anterior movement of the hypoblast (visceral endoderm in mice) lifts its inhibition on the posterior epiblast, allowing NODAL signaling activation. However, in birds, a precise description of this movement was lacking.