Actin polymerization drives lumen formation in a human epiblast model

Dhiraj Indana, Andrei Zakharov, Youngbin Lim, Alexander R. Dunn, Nidhi Bhutani, Vivek B. Shenoy, Ovijit Chaudhuri

Preprint posted on 20 April 2023 https://www.biorxiv.org/content/10.1101/2023.04.20.537711v1#:~:text=Post%2Dimplantation%2C%20the%20pluripotent%20epiblast,human%20epiblast%20lumenogenesis%20are%20unknown

The force drives the lumen of humans: how the actin cytoskeleton generates forces to open the human epiblast lumen.

Selected by Megane Rayer, Rivka Shapiro

Categories: biophysics, cell biology, developmental biology

Background

What we know about lumens?
Many organs feature a lumen, including mammary glands, bile canaliculi, kidneys, salivary glands, etc. A lumen is a hollow cavity surrounded by epithelium, facing the apical surface. Its formation and maintenance are crucial for the proper functioning of organs (for example it allows the release of substances or transport of blood, air and food). Different studies have tried to understand how cells and tissues can form and maintain hollow lumens. There are different mechanisms in which lumens can be created [1]. One of these involves apoptosis: cells in the middle of the glandular epithelium die leaving behind a hollow lumen [2]. Another mechanism involves pressure, specifically an osmotic pressure gradient which applies a compressive force on cells to open and expand the lumen [3, 4].

The beginning of life includes lumen formation.
Without doubt the most important lumen formation process takes place very early during human embryogenesis. During blastocyst implantation, the pluripotent epiblast cells form a lumen which is essential for the rest of embryonic development [5, 6]. However, the mechanism supporting epiblast lumenogenesis remains unknown.

A physical mechanism supporting lumenogenesis.
In this preprint, the authors make clever use of an in vitro human epiblast model to study lumenogenesis. They then propose a mechanism where the first step of epiblast lumenogenesis is the formation of an actin network on the apical side of the cell, increasing cell apical area and driving lumen opening. Then, when the lumen is at a 12 μm average radius, the system switches to the osmotic pressure mechanism.

Key findings

The force driving lumen growth: An apical actin mesh opens the lumen in the early epiblast.

First, the authors could show that apoptosis and osmotic pressure are not involved in early epiblast lumenogenesis, suggesting that there is another mechanism that opens the lumen of the human epiblast.
Knowing that the epiblast model in vitro as well as the in vivo epiblast is constrained by external forces, the authors proposed that the cells themselves need to generate forces to be able to form a lumen, if apoptosis and osmotic pressure are not able to do so. However, treatment with different actomyosin inhibitors didn’t perturb lumenogenesis, which led the authors to conclude that a contractile force generating machinery is not required in this system.
However, the actin cytoskeleton densely localized to the apical side of the epiblast. The authors therefore wondered how this apical actin is organized and if it can generate forces. Surprisingly, and thanks to high resolution confocal microscopy, the authors were able to distinguish, among this dense actin accumulation, both microvilli and an apical meshwork. Interestingly this apical meshwork was enriched in N-wasp and arp2/3, suggesting the formation of a branched actin network. To test if the actin polymerization could drive epiblast lumenogenesis, the authors used arp2/3, formins, and N-wasp chemical inhibitors; all of these drugs prevented lumen formation. However, after 7 days of culture the drugs had no effect on lumen growth, showing that actin polymerization is only necessary to open the lumen up in the very early epiblast. Actin polymerization in individual cells correlated with apical surface expansion which reached an equilibrium size at a lumen radius of 12 μm.
But how can actin polymerization drive lumen opening? The authors answered this question through live imaging, laser ablation experiments, quantification of hydrogel deformation and computational modeling. They showed that in a smaller epiblast, actin polymerization is a single cell dependent process that exerts a planar apical pushing force which increases the size of the apical surface. They also showed that the apical forces promote the resistance to extracellular matrix pressure.

Pressure extends the lumen: osmotic pressure allows lumen growth.

The authors next investigated which mechanisms are involved in maintaining and growing lumens larger than 12 μm in radius where actin polymerization stalls. In these larger epiblasts, functional tight junctions form, suggesting a potential buildup of osmotic pressure inside the lumen that can push the cells. They tested this hypothesis by laser ablating the apical surface, showing that the cells are under tension to resist the luminal pressure. Interestingly, their computational model demonstrated that ion pumping builds osmotic pressure and supports robust lumen growth, with a lumen size close to the experimental observations. Furthermore, perturbing apico-basal ion pumping prevented lumen growth, strongly showing that the osmotic pressure allows lumen expansion in epiblasts with lumens > 12μm radius.

Our opinion.

Understanding lumen morphogenesis is crucial for understanding development itself, as described in this study. However, it is also very important for understanding cancer progression. Indeed, the loss of the lumen in glandular epithelium is a hallmark of cancer. Here, the authors offer a real advance in our understanding of human embryonic development as a whole, as well as improve our knowledge of lumen formation and physiology.
This preprint is fascinating because it shows that individual cells can generate forces to form a lumen. The death of cells and/or osmotic pressure are not sufficient to initiate lumenogenesis, suggesting the existence of many other mechanisms. This knowledge is very useful in our own research on lumen formation and maintenance in mammary glands to better understand the development of breast cancer.

Questions for the authors:

Q1. As you have shown in your study, myosin is mostly located at the basal side. Do you think this specific location allows the tissue to resist the extracellular matrix pressure during lumenogenesis? What could be the role of this subcellular localization?

Q2. You have demonstrated that while the apical side grows, overall cell volume stays constant. In order for this to be the case, another part of the cell must be shrinking. Do you think that this could be the basal membrane? Did you observe any other phenomena at the basal side which contrast to what occurs on the apical side?

Q3. What triggers the tight junctions to form consistently in larger epiblasts? Can the epiblasts sense their own size? How do you think this mechanism might work?

References.

1. Sigurbjörnsdóttir, S., R. Mathew, and M. Leptin, Molecular mechanisms of de novo lumen formation. Nat Rev Mol Cell Biol, 2014. 15(10): p. 665-76.
2. Debnath, J., et al., The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell, 2002. 111(1): p. 29-40.
3. Chan, C.J. and T. Hiiragi, Integration of luminal pressure and signalling in tissue self-organization. Development, 2020. 147(5).
4. Torres-Sánchez, A., M. Kerr Winter, and G. Salbreux, Tissue hydraulics: Physics of lumen formation and interaction. Cells Dev, 2021. 168: p. 203724.
5. Ryan, A.Q., et al., Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation. Dev Cell, 2019. 51(6): p. 684-697.e4.
6. Shahbazi, M.N. and M. Zernicka-Goetz, Deconstructing and reconstructing the mouse and human early embryo. Nat Cell Biol, 2018. 20(8): p. 878-887.
7. Fu, J., A. Warmflash, and M.P. Lutolf, Stem-cell-based embryo models for fundamental research and translation. Nat Mater, 2021. 20(2): p. 132-144.
8. Bao, M., J. Cornwall-Scoones, and M. Zernicka-Goetz, Stem-cell-based human and mouse embryo models. Curr Opin Genet Dev, 2022. 76: p. 101970.

Tags: biophysics, human development, in vitro embryo, lumen formation

Posted on: 5 April 2024

doi: https://doi.org/10.1242/prelights.37021

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Author's response

Dhiraj Indana shared

A1. Yes, we observe that myosin is mostly located at the basal surface, but the localization is sparse. We believe that myosin dependent contractile forces do not play a major role during lumenogenesis in our epiblast model, in contrast to Drosophila gastrulation for example. However, basally localized myosin could help resist some extracellular matrix pressure though we haven’t tested this hypothesis.

A2. Yes, exactly. We see that as cells polarize while concomitantly expanding their lumen, apical and basal domains emerge. We observe that cells maintain nearly constant volume and total surface area. So as the apical area grows till an equilibrium size of around 100 square microns, basal surface shrinks in area.
In addition to differences in myosin localization between the apical and basal surfaces, we observe previously reported differences including localization of actin, podocalyxin, ezrin and tight junctions at the apical surface.

A3. Excellent question! How the mechanism of lumen growth switches from actin polymerization to osmotic pressure is a mystery. We see that at a lumen radius of around 12 microns, cells form tight junctions and also reach an equilibrium apical size. It is currently unclear how these two processes are correlated and what triggers these events. Do cells sense the lumen size or number of cells or their own shapes? These are all open questions and we hope to uncover this in the future.

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Actin polymerization drives lumen formation in a human epiblast model

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