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Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues

Guillermo Martínez-Ara, Núria Taberner, Mami Takayama, Elissavet Sandaltzopoulou, Casandra E. Villava, Nozomu Takata, Mototsugu Eiraku, Miki Ebisuya

Preprint posted on April 21, 2021 https://www.biorxiv.org/content/10.1101/2021.04.20.440475v1

Bending with light: OptoShroom3 an optogenetic toolbox to trigger 3D tissue deformation and control morphogenesis

Selected by Monica Tambalo

Background

A fundamental question that has always fascinated scientists is how complex shapes emerge during embryo development to give rise to complex tissues/organs. Morphogenesis is the highly controlled process by which coordinated changes in cell behaviors (e.g., changes in cell shape, remodeling of cell contacts, cell migration, cell division and cell extrusion) lead to the formation of 3D tissues and organs. Apical constriction is a mechanism that is widely used during development to modulate epithelial tissue curvature. The constriction of the apical surface of a cell produces cell shape changes, which, when coordinated across neighboring cells, leads to overall tissue bending. Actomyosin contraction is the driving force of apical constriction, which is frequently caused by apical activation of the Rho-ROCK pathway. A great overview of this process has been recently reviewed using Drosophila as an example (Perez-Vale and Peifer, 2020). During development, apical constriction is tightly regulated: it occurs in focal areas (space control) and at specific time points (time control). In this recently published preprint Martínez-Ara and colleagues (Martinez-Ara et al., 2021) have developed an optogenetic toolbox to manipulate apical constriction and investigate morphogenesis in a spatio-temporally precise way.

In brief, optogenetics uses light-based control of protein domains (photo-receptors) which undergo conformational changes upon exposure to specific wavelengths (Tischer and Weiner, 2014; Liu and Tucker, 2017). An array of optogenetic tools are available for scientists to challenge morphogenesis (Hartmann et al., 2020; Mumford et al., 2020). Of relevance for this work, Izquierdo and colleagues have previously developed an optogenetic tool to recruit RhoGEF to the plasma membrane of Drosophila cells which led to tissue invagination upon light-activation (Izquierdo et al., 2018).

The authors of this preprint focused on the development of a cutting-edge optogenetic tool to precisely manipulate mammalian morphogenesis, focusing on the great potential for synthetic tissue engineering in stem cell-derived 3D organoids.

Key findings

To generate a suitable optogenetic tool the authors took advantage of a light inducible protein dimerization system based on an improved light inducible dimer (iLID) which, upon blue light stimulation, is able to bind to its natural binding partner (SspB peptide) to form a light-induced dimer (Guntas et al., 2015). This is a particularly powerful tool because it can be used to localize and activate proteins in a controllable manner. In this preprint the authors developed an optogenetic version of Shroom3, a key regulator of apical constriction by ROCK recruitment, to achieve spatiotemporal control of morphogenesis in mammalian tissues. Shroom3 is composed of two independent domains that were separated into two plasmid constructs, Shroom3 optogenetic tool (OptoShroom3) was designed as follows (Fig 1A):

i) N-terminal – Shroom Domain 1 (SD1) the actin-binding motif responsible for the apical localization – fused with iLID (NShroom3-iLID),

ii) C-terminal – Shroom Domain 2 (SD3) necessary for ROCK binding – fused with SspB (SspB-CShroom3).

Figure 1. OptoShroom3-induced apical constriction in several mammalian cell contexts.
A) Design of OptoShroom3 constructs that dimerize upon blue light stimulation. B) Flat MDCK monolayers generation and colony folding after 24 h of OptoShroom3 stimulation. C) Stimulation cycles of mouse optic vesicle organoids causes tissue shrinkage. D) OptoShroom3 activation in human neuroectodermal organoids causes neuroectoderm flattening.

To test the functionality of OptoShroom3, a single cell in a Madin-Darby Canine Kidney (MDCK) monolayer was stimulated with light. A 1-minute illumination cycle was firstly tested showing a rapid decrease in the apical surface with a substantial reduction in area over 50 minutes stimulation. Importantly, 1 minute after the end of the stimulation the apical surface started to increase again, thus demonstrating a fast dynamic of the system. OptoShroom3 can also be repeatedly activated and deactivated by performing several stimulation-rest periods. Thus, OptoShroom3 was proven to be a suitable tool to temporally control apical constriction with a fast activation and deactivation kinetics.

To manipulate apical constriction at the tissue-level a larger area was stimulated. A MDCK monolayer, grown on a collagen gel permissive to deformations, was photostimulated. This caused cellular displacement towards the stimulated area on the apical side, followed by displacement in the opposite direction after the end of stimulation. Additionally, more complex patterns of stimulation were performed in 2D. These sets of experiments have shown that OptoShroom3 can induce 2D tissue-level deformations by inducing apical constriction on cells adjacent to the stimulated area.

MCDK cells were then grown on Matrigel (treated with acetic acid to enable growth of flat MDCK monolayer colonies) to test 3D tissue morphogenesis. Remarkably, 24 hour photostimulation of the whole colony led to monolayer folding, resembling mammalian folding processes (e.g. neural tube and gut folding) (Fig. 1B). This experiment together with few other variations led the authors to conclude that OptoShroom3 can induce tissue curvatures and folding.

To study morphogenesis in complex 3D mammalian tissues the authors generated a stable mouse embryonic stem (mES) cell line expressing OptoShroom3, which was used to generate optic vesicle organoids. The authors chose optic vesicle organoids since it has been demonstrated that apical constriction is fundamental for eye morphogenesis (Eiraku et al., 2011; Okuda et al., 2018). In this system GFP-NShroom3-iLID has a dynamic localization, appearing on the apical side of the neuroepithelium (inner surface of optic vesicle) after 4 days of in vitro culture. Photostimulation of OptoShroom3 at 4-8 days caused an increase in the thickness of the epithelial layer, while later stimulation (days 6-8) caused an overall decrease in the apical lumen (Fig. 1C). This set of experiments point to the conclusion that OptoShroom3 can affect cell shape, and by inducing apical constriction, leads to apical lumen reduction.

As a final approach the authors tested their toolbox on human neuroectodermal organoids. Human induced pluripotent stem cells (hiPSC) stably expressing OptoShroom3 were used to generate neuroectodermal organoids. Importantly, neuroectodermal organoids present a convex apical side, with outer apical polarity. Photostimulation stimulation of the neuroectoderm border led to a flattening of the area causing inward tissue bending (Fig. 1D).

The great achievement of this study is the development of a versatile optogenetic toolbox that functions in multiple contexts. Of relevance is that this novel tool can specifically promote OptoShroom3 recruitment to the apical junctions of epithelial cells, not requiring precision light stimulation (multi-photon microscopy) as needed for other tools. Thanks to such unique features OptoShroom3 can be elegantly used to control tissue shapes and further investigate mechanisms of tissue morphogenesis. OptoShroom3 also allows for fine temporal control of the deformation due to rapid activation-deactivation cycle, but if needed long-term stimulations are also possible. Another take-home message is that light deformation is dependent on the original tissue geometry (apicobasal polarity, mechanical forces in place, and availability of cytoskeletal components).

Why I like this preprint

I am fascinated by how complex structures emerge during development, and I find OptoShroom3 a new versatile tool to further learn the role of morphogenesis. In my opinion, this tool is extremely useful and has great potential in many different contexts. As mentioned in the discussion, organoids are a great system for studying morphogenesis and investigating how shape changes impact cell fate and differentiation. I find such future avenues of great biological interest and I look forward to seeing the applications of OptoShroom3 in future studies!

Questions to the authors

Q1) Do you have any advice for researchers embarking on an optogenetic project (design/generation of tools, in vivo/in vitro testing, time required for experiment set up)?

Q2) For how long after photostimulation were you able to see apical contraction and tissue deformation? Would this tool or a variation of it allow to permanently modify tissue shape?

Q3) How do you envisage this tool to be used in the near future?

References

Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, K., Adachi, T., Sasai, Y., 2011. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–58. https://doi.org/10.1038/nature09941

Guntas, G., Hallett, R.A., Zimmerman, S.P., Williams, T., Yumerefendi, H., Bear, J.E., Kuhlman, B., 2015. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl. Acad. Sci. 112, 112–117. https://doi.org/10.1073/PNAS.1417910112

Hartmann, J., Krueger, D., De Renzis, S., 2020. Using optogenetics to tackle systems-level questions of multicellular morphogenesis. Curr. Opin. Cell Biol. 66, 19–27. https://doi.org/10.1016/j.ceb.2020.04.004

Izquierdo, E., Quinkler, T., De Renzis, S., 2018. Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis. Nat. Commun. 9, 1–13. https://doi.org/10.1038/s41467-018-04754-z

Liu, Q., Tucker, C.L., 2017. Engineering genetically-encoded tools for optogenetic control of protein activity. Curr. Opin. Chem. Biol. 40, 17–23. https://doi.org/10.1016/j.cbpa.2017.05.001

Martinez-Ara, G., Taberner, N., Takayama, M., Sandaltzopoulou, E., Villava, C.E., Takata, N., Eiraku, M., Ebisuya, M., 2021. Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues. bioRxiv 2021.04.20.440475.

Mumford, T.R., Roth, L., Bugaj, L.J., 2020. Reverse and forward engineering multicellular structures with optogenetics. Curr. Opin. Biomed. Eng. 16, 61–71. https://doi.org/10.1016/j.cobme.2020.100250

Okuda, S., Takata, N., Hasegawa, Y., Kawada, M., Inoue, Y., Adachi, T., Sasai, Y., Eiraku, M., 2018. Strain-triggered mechanical feedback in self-organizing optic-cup morphogenesis. Sci. Adv. 4, 1–13. https://doi.org/10.1126/sciadv.aau1354

Perez-Vale, K.Z., Peifer, M., 2020. Orchestrating morphogenesis: Building the body plan by cell shape changes and movements. Dev. 147, 1–16. https://doi.org/10.1242/dev.191049

Tischer, D., Weiner, O.D., 2014. Illuminating cell signalling with optogenetic tools. Nat. Rev. Mol. Cell Biol. 15, 551–558. https://doi.org/10.1038/nrm3837

Tags: morphogenesis, optogenetic, tissue engineering

Posted on: 8th July 2021

doi: https://doi.org/10.1242/prelights.29952

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Author's response

Guillermo Martínez-Ara & Miki Ebisuya shared

Q1) Do you have any advice for researchers embarking on an optogenetic project (design/generation of tools, in vivo/in vitro testing, time required for experiment set up)?

In general, it’s very difficult to predict whether an optogenetic construct will work or not. For this reason, we recommend to dedicate some time at the beginning of the project to build many alternative constructs to increase the chances of success and save time in the design stage. By changing the order of the components, linkers, and trying different optogenetic systems, they will be more likely to find a functional optogenetic tool without spending too much time.
Once the constructs are built, it can take some time to find the optimal range of parameters that make the tool function. Sometimes we may believe a construct does not work properly, but maybe it is just not being stimulated properly. Checking the literature on previous tools using the same optogenetic components (iLID, Cry2, PhyB…) can help a lot with this.

Q2) For how long after photostimulation were you able to see apical contraction and tissue deformation?

Would this tool or a variation of it allow to permanently modify tissue shape?
In general, we observe that contractility ends about 1 minute after stimulation stops. In the case of short periods of stimulation (less than one hour), this means that cells relax and go back to their original shapes. We are currently investigating if longer stimulation periods that allow for cell division and cell-cell rearrangements to happen may lead to more stable changes in tissue shape.

Q3) How do you envisage this tool to be used in the near future?

We hope OptoShroom3 will be helpful in multiple fields, from the study of cytoskeletal dynamics, to probing forces on epithelial monolayers, and also for the exploration of morphogenesis on in vitro and in vivo systems. In our lab, we are especially excited about studying the interaction between tissue shape, function and differentiation.

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