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Optogenetic control of Wnt signaling for modeling early embryogenic patterning with human pluripotent stem cells

Nicole A. Repina, Xiaoping Bao, Joshua A. Zimmermann, David A. Joy, Ravi S. Kane, David V. Schaffer

Preprint posted on June 10, 2019 https://www.biorxiv.org/content/10.1101/665695v1

Stem cells see the light! Controlling WNT signaling using optogenetics to interrogate self-organization in stem cells.

Selected by Pavithran Ravindran

Background

Cells are constantly bombarded with extracellular ligands and to encode this information they activate signaling pathways in distinct time-varying patterns. How such signals are interpreted in order to make cell fate decisions is largely unclear1,2. The ability to directly test how such signals are interpreted hinges on ones ability to precisely control particular signaling pathways using experimental methods. Optogenetics, the use of light responsive proteins to recruit signaling pathway effectors, has begun to play an essential role in filling this need. Work has been done to control particular pathways in cells, zebrafish and Drosophila embryos3. In this work, the authors decide to further their previously published optogenetic tool to control WNT signaling by integrating the system into stem cells4.

Key Findings

In this preprint, the authors set out to develop a method to study stem cells to study how spatial and temporal signaling dynamics regulate cell fates. Classical methods in which scientists use exogenous ligands or inhibitors to study signaling pathways gives poorly defined spatial resolution (every cell is treated the same) and temporal resolution (it is difficult to toggle a pathway on and off). To overcome this issue, the authors use a previously published optogenetic tool that specifically activates the WNT signaling pathway by using a blue-light inducible clustering system known as Cry2 attached to the WNT responsive receptor LRP6, what they called the OptoWNT system (figure 1). After integrating the OptoWNT system into the AAVS1 safe harbor locus of human embryonic stem cells (hESCs), the authors validate that blue light causes reversible clustering of LRP6, and accumulation of the Wnt transcriptional activator beta-catenin. From there, the authors wanted to ensure that Wnt activation through optogenetics in these stem cells recapitulates the known differentiation after recombinant Wnt3a activation. By comparing levels of Brachyury, a master mesendoderm transcription factor and marker of the primitive streak, in cells that got no light or constant 48 hours of blue light exposure, the authors found that there was a 40 fold increase in protein expression with blue light. Finally, by performing RNA-sequencing on both WT hESCs and hESCs integrated with the OptoWNT system in dark and blue light conditions, the authors found that there was very little photo-toxicity from continuous blue light exposure, low OptoWNT dark state activity and induction of differentiation upon blue light stimulation. All of these results suggest that the optogenetic tool for WNT activation integrated into the AAVS1 locus functions in a blue light dependent manner.

Figure 1: Schematic of optogenetic system to control WNT signaling in stem cells. Cry2 is attached to the cytoplasmic portion of WNT receptor LRP6. Upon blue light stimulation, LRP6 clusters and causes an accumulation of β-catenin. Taken from Figure 1A of Repina et al, preprint.

 

Once the authors were able to validate the OptoWNT system in hESCs, they wanted to address a question that classic drug/growth factor additions could not: what effect does the activation of WNT have when only a subset of cells in a population are “listening” for this activation? To answer this, they mixed wild-type hESCs and hESCs with OptoWNT (which were also mCherry positive such that they could distinguish between the two populations) and then applied blue light. They found that upon this blue light stimulation, the mCherry OptoWNT hESCs would segregate out and thus would have more mCherry positive neighbors as opposed to WT neighbors. They found that this segregation occurred in almost any media they checked, including media without FGF2 and TGFβ, suggesting that WNT activation alone was sufficient to drive this self-organization. Even more impressively, the authors found that self-organization occurred in a 3D co-culture of wild-type and OptoWNT hESCs in which the OptoWNT activated cells formed a ring around the wild-type cells (figure 2). All of these results suggest that WNT activation in a subset of cells may be sufficient to drive self-organization of cells.

Figure 2: Schematic of co-culture experiment between OptoWNT and wildtype hESCs. After blue light stimulation, optoWNT hESCs differentiate and move to the outside of the spheroid. Taken from figure 4E of Repina et al, preprint.

 

Why I chose this preprint

Optogenetics is an amazing tool that allows researchers spatiotemporal control of signaling pathway activation in cells. Sadly however, the tool has been used mostly in either immortalized cell lines, which are far from normal biological representatives, or full organisms such as the embryo, which take a long time to develop. This work seems to hit the sweet spot directly in between these extremes by applying optogenetics to an interesting biological system that also has the advantages of normal cell culture. With this work, it will be extremely interesting to see what biologists will be able to learn about stem cells and the paths they take towards differentiation.

Questions for the authors

  1. In this work you have done a great job is showcasing the utility of optogenetics for the selective activation of a subset of cells within a population. Another great use of this tool is in the temporal dimension. Were you able to apply dynamic pulses of blue light to these cells and did this have any effect on the self-organization that you have described in your work?
  2. You have proposed a model in which WNT activation in a subset of cells is sufficient to drive self-organization, possibly for a developmental case. Do you know of any cases in which this may occur in the early embryo? Do you have a hypothesis as to the mechanism of only certain cells being receptive to the input?

References 

  1. Purvis, J. E. & Lahav, G. Encoding and decoding cellular information through signaling dynamics. Cell 152, 945–956 (2013).
  2. Maryu, G. et al. Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters. 74, 61–74 (2018).
  3. Johnson, H. E. & Toettcher, J. E. Illuminating developmental biology with cellular optogenetics. Curr. Opin. Biotechnol. 52, 42–48 (2018).
  4. Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Optogenetic protein clustering and signaling activation in mammalian cells. 10, 249–252 (2013).

 

Posted on: 30th July 2019 , updated on: 23rd August 2019

Read preprint (1 votes)




  • Author's response

    Nicole Repina shared

    1) Temporal modulation of a signal is indeed one of the great advantages of an optogenetic tool. Given the clustering and dissociation kinetics of Cry2, the optoWnt system can be used to deliver oscillatory signals, with a shortest achievable period of ~30 min [1,2]. In our work, we did not apply an oscillatory signal to optoWnt/WT cell co-cultures since we are modeling the process of primitive streak formation, where such oscillatory signals within the embryo have not been found. However, in later stages of development, temporal oscillations of Wnt regulate morphogenesis, such as during mesoderm segmentation and somitogenesis [3]. It would thus be interesting to use the optoWnt system to, for example, interrogate signal entrainment and frequency response during such developmental stages.

    2) Wnt activation in a subset of cells is one of the hallmarks of early embryogenesis. Prior to primitive streak formation, a gradient of Wnt signaling emerges across the mouse epiblast. The gradient is reinforced by a Nodal/BMP/Wnt signaling feedback loop between the epiblast and extraembryonic ectoderm tissue [4], as well as by inhibitory signaling hubs that form in the distal and, subsequently, anterior visceral endoderm. The origin of signal asymmetry, however, remains unclear, and may arise from a localized Wnt morphogen secretion from the proximo-posterior visceral endoderm [5]. However, conditional ablation of Wnt3 in the visceral endoderm delayed but did not inhibit axis formation and gastrulation [6], suggesting that the multiple signaling mechanisms patterning the epiblast may have compensatory effects. For example, cell-to-cell heterogeneity within the epiblast may play a role, where certain cells are more responsive to a Wnt stimulus or initiate patterning through stochastic fluctuations in gene expression [7]. The mechanism by which only certain cells receive or are receptive to a Wnt stimulus thus remains unclear, and may indeed be a result of multiple factors. Our work models the case where Wnt signaling is stimulated heterogeneously in a subset of cells to show that Wnt is sufficient for cell self-organization and morphogenesis.

    1. Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Nat. Methods 10, 249–252 (2013)
    2. Repina, N. A., McClave, T., Bao, X., Kane, R. S. & Schaffer, D. V. bioRxiv 8, 675892 (2019)
    3. Sonnen, K. F. et al. Cell 172, 1079–1090.e12 (2018)
    4. Ben-Haim, N. et al. Dev. Cell 11, 313–323 (2006)
    5. Rivera-Perez, J. A., Mager, J. & Magnuson, T. Developmental Biology 261, 470–487 (2003)
    6. Yoon, Y.et al. Developmental Biology 403, 80–88 (2015)
    7. Wennekamp, S., Mesecke, S., Nedelec, F. & Hiiragi, T. Nat. Rev. Mol. Cell Biol. 14, 452–459 (2013)

    Thank you again for the wonderful prelight!

    All the best,

    Nicole

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