Live imaging and functional characterization of the avian hypoblast redefine the mechanisms of primitive streak induction

Introduction

The textbook model of primitive streak induction is an interplay between the hypoblast and the epiblast, the two cell layers of the early amniote embryo: initially, the hypoblast secretes antagonists of NODAL and WNT to inhibit primitive streak formation in the epiblast; later, the hypoblast becomes displaced anteriorly, thus permitting the streak to initiate in the posterior epiblast. Such a mechanism could explain how the primitive streak is positioned at the posterior, and how the embryo ensures only one streak is formed.

However, while this model is well-supported by experimental evidence in mice, whether it is the case in the avian embryo remains unclear. In support of the model, it has been reported that expression of hypoblast genes, which include inhibitors of NODAL and WNT, is progressively relocated to the anterior; also, ablation of the hypoblast can occasionally produce ectopic primitive streaks. However, the anterior motion of the hypoblast, which is the central part of the model, has only been inferred from poorly resolved imaging and tracing of sparsely labelled cells. In particular, the proposed relative displacement between hypoblast and epiblast was never directly observed.

Luckily, transgenic lines and fluorescence imaging now allow us to actually see tissue-scale morphogenetic movements at cell resolution. In this preprint, Aurélien Villedieu and colleagues live-imaged avian blastoderm tissue flows. What they found in the hypoblast was not the expected anterior movement, but rather a passive rotation driven by the epiblast. Through detailed analysis of gene expression dynamics and tissue grafting assays, authors further propose that avian primitive streak formation is induced, rather than inhibited by the posterior hypoblast.

Main findings

1. Counter-rotating flows in the hypoblast

Contrary to expectation, cell-resolution live imaging of the hypoblast showed a large-scale, counter-rotating tissue flow, similar to the Polonaise movement in the epiblast during primitive streak formation.

2. The epiblast entrains and deforms the hypoblast during primitive streak formation

Photoconversion labelling showed that hypoblast and epiblast cells remained close together. Simultaneously recording both hypoblast and epiblast movements also showed little relative motion.

Inserting a porous filter between the hypoblast and the epiblast, which allowed exchange of diffusible molecules but blocked mechanical contact, would abolish the counter-rotating flows in the hypoblast.

The hypoblast also did not show detectable levels of junctional phosphorylated Myosin II, which argues against active migration of hypoblast cells.

Together, authors concluded that the rotational movement of the hypoblast is likely a passive deformation resulting from epiblast movement.

3. NODAL expression initiates in the posterior hypoblast and propagates in correlation with the progressive patterning of the hypoblast.

Using HCR-RNA-FISH, NODAL expression was first detected in the posterior hypoblast, and later also in posterior epiblast, forming a posterior-to-anterior gradient in both layers. GDF1 expression was detected later than NODAL, starting in the epiblast, then in the hypoblast more anteriorly than NODAL.

As previously reported, the early hypoblast marker FOXA2 changed from a homogeneous field to an anterior-to-posterior gradient, opposite to that of NODAL. The posterior high-NODAL/low-FOXA2 domain was traced back to the transition between posterior hypoblast and central hypoblast islands, a region that would have been highly expressing FOXA2, and also where NODAL expression emerged.

Authors thus argued that the FOXA2 gradient is formed by cells downregulating FOXA2 expression in response to genetic regulation, rather than cells being displaced by a different low-FOXA2 cell population.

4. NODAL signaling in the posterior hypoblast induces the primitive streak.

Surgical removal of hypoblast cells either abolished or compromised primitive streak formation and epiblast NODAL expression.

Interestingly, when two pieces of posterior hypoblast were left un-removed on each side of the ablated zone, the epiblast would form either 2 primitive streaks facing the untouched hypoblast, or a single primitive streak at a shifted location. Authors thus argued that the previous result that surgical ablation of the hypoblast produces additional axes was in fact due to incomplete hypoblast removal.

A graft of posterior hypoblast or an Activin A (NODAL surrogate) bead to the anterior would induce an ectopic primary axis, and induce FOXA2 decrease and NODAL activation in surrounding hypoblast tissue. Conversely, treating the whole embryo with Nodal inhibitor SB505124 would prevent primitive streak formation, and also prevent FOXA2 downregulation and CER1 expression in the hypoblast.

Together, authors concluded that Nodal activity in posterior hypoblast is both required and sufficient to trigger primitive streak formation and hypoblast transcriptional regulations.

5. The anterior hypoblast is unable to inhibit primitive streak formation

Expression of NODAL inhibitor CER1 was detected in anterior hypoblast, but it emerged later than NODAL patterning and primitive streak initiation, thus CER1 is unlikely to regulate NODAL patterning.

Tissue grafting assays showed that the anterior hypoblast could not inhibit epiblast NODAL expression, regardless of whether CER1 expression is present. Instead, the hypoblast grafted would upregulate NODAL and downregulate FOXA2, appearing to be converted to a posterior identity. This might be attributed to NODAL-expressing posterior epiblast, which could induce an ectopic streak, and also hypoblast NODAL expression and FOXA2 downregulation in the host hypoblast.

Together, authors proposed that there is a positive feedback loop between the epiblast and the hypoblast. The posterior hypoblast can induce NODAL expression and primitive streak formation in the epiblast, and the epiblast would maintain NODAL expression and downregulate FOXA2 expression in the hypoblast.

 

What I like about this preprint

What really drew me to this preprint is how it puts the long-standing model of primitive streak formation to the test—by actually watching tissue flows in real time at high resolution. The comprehensive view of how tissues move provided compelling pieces of direct evidence, allowing authors to challenge the old model on firm grounds. I look forward to seeing more studies that bring vertebrate morphogenesis to life in real time.

 

Questions for authors

1. Does the counter-rotating flow in the hypoblast have a biological role? In particular, as the flow displaces cells, changes neighbouring relationships and potentially presents mechanical cues, how do you think it would affect hypoblast patterning by molecular signals?
2. Can you expound more on the mechanical details of how epiblast flows could drive such large-scale deformations/cell displacements in the hypoblast? Also, how would this tight mechanical coupling still allow for the intrinsic active expansion of the hypoblast?

 

doi: https://doi.org/10.1242/prelights.40995

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