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Self-organised symmetry breaking in zebrafish reveals feedback from morphogenesis to pattern formation

Vikas Trivedi, Timothy Fulton, Andrea Attardi, Kerim Anlas, Chaitanya Dingare, Alfonso Martinez-Arias, Benjamin Steventon

Preprint posted on September 18, 2019 https://www.biorxiv.org/content/10.1101/769257v2

Pescoids - a new in vitro model system to study early zebrafish embryogenesis

Selected by Sundar Naganathan

Categories: developmental biology

Background

Morphogenesis is the process by which groups of cells transform in to tissues of diverse shapes and forms through a complex interplay between gene expression patterns and mechanical processes. To understand morphogenesis, several model systems have been successfully used to document and characterize gene expression patterns spatiotemporally. Yet we are still a long way from understanding the mechanisms by which intricate patterns emerge in embryos in a robust manner. Do early embryos have an intrinsic ability to self-organize? If yes, what are the principles of this self-organization process? Are extra-embryonic tissues, such as the yolk syncytial layer in zebrafish embryos and trophectoderm in mouse embryos important in regulating spatiotemporal gene expression patterning?

Several reports have established that extra-embryonic tissues do play a role in driving morphogenetic events such as mesodermal induction and epiboly in zebrafish1 and anteroposterior and dorsoventral axes establishment in mouse embryos2. However, the advent of the gastruloid model system3, which emerges from mouse embryonic stem cells, has provided an alternative view of early symmetry breaking, where many morphogenetic events such as elongation and anteroposterior patterning emerge in the absence of extra-embryonic tissues. The authors of this preprint further stimulate this debate, by developing a gastruloid-like primary culture system using zebrafish early embryonic cells.

Key observations

The authors explanted zebrafish embryonic cells from the 256-cell stage (~2.5 hrs post fertilization) and characterized emergent morphogenesis in the explants. The explanted cells aggregated and underwent rapid rounds of cell division before elongating from one end. More than 60% of the explants exhibited this polarization suggesting that symmetry breaking can occur spontaneously in embryonic cells in the absence of signals emanating from the yolk. Interestingly, symmetry breaking was observed even when explanted cells were dissociated and re-aggregated. This experiment, in addition to the observation of homogeneous cell mixing upon explantation, suggests that symmetry breaking is likely to be an intrinsic ability of early embryonic cells and is independent of pre-patterns preserved from the embryo.

The self-organized cell aggregates were named pescoids, which exhibited the following spatial gene expression patterns, in addition to the morphological symmetry breaking event:

  1. Mesodermal marker expression in the elongating end;
  2. High BMP activity in a region opposite to the elongating end;
  3. Inhibitors of BMP signalling in the elongating end;
  4. Spatially distinct expression of anteroposterior neural patterning genes, with the posterior genes closer to the elongating end

In vivo, the yolk plays a major role in breaking initial symmetries in the embryo. Do vegetal cells then, which are spatially closer to the yolk, display an enhanced ability to self-organize in explants? To test this, the authors explanted animal and vegetal cells separately and observed that both explants were equally capable of exhibiting elongation. However, the elongation length was smaller than that observed in pescoids suggesting that both animal and vegetal regions are necessary for elongation. Interestingly, when the sizes of the animal and vegetal explants were increased by increasing the starting number of cells, the ability to elongate increased. This suggests that tissue size and large-scale mechanics of the entire aggregate could play a role in determining elongation.

What regulates elongation in the pescoids? The authors observed that spatially localized Nodal signalling precedes mesodermal marker expression and elongation, both of which were abolished upon inhibition of nodal signalling. These observations agree well with recent work where in-depth characterization of Nodal signalling during convergence extension was performed4. Furthermore, inhibition of the planar cell polarity pathway, a major regulator of convergence-extension movements in vivo, also blocked pescoid elongation and severely affected anteroposterior patterning. This suggests that a combination of self-organized Nodal signalling and convergence-extension movements drive pescoid elongation and patterning.

Why I chose this preprint?

The authors have established a new in vitro model system for analysis of early zebrafish embryo morphogenesis. This enables quantitative comparisons to analysis performed in intact embryos providing important information on the role of extra-embryonic tissues in regulating morphogenesis. Furthermore, any in vitro system brings with it an advantage of performing controlled perturbations and pescoids is no different in this respect.

The study has uncovered that early embryonic cells have an intrinsic ability to break symmetry and this discovery is likely to spur in-depth quantitative analysis of symmetry breaking processes and downstream self-organization of spatial gene expression patterns, which are not well understood in vertebrate model systems.

Open questions

  1. The same culture dish seems to have many pescoids at the same time. If a pescoid is cultured in isolation, do the authors expect any change in the quantified parameters? In other words, do pescoids influence each other through long-range signalling?
  2. 3% FBS has been used to culture the pescoids. FBS has a range of different growth factors such as FGF and TGF-b1, which may affect the observed morphogenesis. It is therefore tricky to interpret the observed dynamics and necessitates further control experiments to test the impact of FBS on pescoid morphogenesis.
  3. Even with an aggregate size similar to that of the pescoids, a similar elongation length was not observed in the animal and vegetal explants. This argues that aggregate size may not be the major determining factor in pescoid elongation in contrast to the interpretation in the preprint. Further experiments are required to clearly understand the importance of tissue size in regulating elongation.
  4. In Fig. 3c left image, it appears that the orientation of cell divisions in the periphery of the pescoid seem to align with local curvature of the pescoid. Could the authors test if the material properties of the pescoid in the periphery are different from the interior, where cell division orientations seem to be more random?
  5. Any ideas on why explants generated from embryos that are younger than the 256-cell stage fail to elongate efficiently?

References

  1. Carvalho L. and Heisenberg C. P., The yolk syncytial layer in early zebrafish development, Trends Cell Biol., 2010
  2. Rivera-Péréz J. A. and Hadjantonakis A-K., The dynamics of morphogenesis in the early mouse embryo, Cold Spr. Har. Persp. Biol., 2015
  3. van den Brink S. C. et al., Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells, Dev., 2014
  4. Williams M. L. K. and Solnica-Krezel L., A mesoderm-independent role for Nodal signaling in convergence & extension gastrulation movements, bioRxiv, 2019

Tags: convergence extension, pattern formation, symmetry breaking

Posted on: 7th October 2019

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  • Author's response

    Benjamin Steventon shared

    1. We have not done this specifically, although we could note that we culture in large volumes (and that this is important for their survival), this suggests that any secreted signals would rapidly diffuse in the medium.
    2. Fig. S5 and S6 in the Supp Info show that with a varying degree of media composition (L15, DMEM, PBS) and FBS % (1%, 3%, 10%), all conditions allow elongation.
    3. The animal and vegetal explants are always smaller than complete pescoids, so this doesn’t rule out a role for explant size being important. However, it would indeed be interesting to do more animal cap fusion experiments to further confirm the importance of aggregate size. In particular, the effect of having extra tissue has not be fully explored.
    4. It is certainly true that the outer part of the explant takes on different gene expression from the inside, in particular Krt8 expression. Exactly when and how inner vs. outer layers formed will be an important thing to explore in the future.
    5. Most likely this is due to the proper aggregation of the cells. The reason could also be due to the fact that till 32 cell stage, all cells are contiguous with the yolk, so we cannot make explants at this stage. As division starts we get more cells to make explants but the error in manually removing cells with eyelash tool is higher at earlier stages (64-stage and 128-stage) as compared to later stages. So it is likely that we start with less number of cells (critical number) if we harvest them from earlier embryos.

     

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