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  1. Evaluation Summary:

    Pond et al. established a 2D patient-derived organoid screening platform to study tissue patterning and kinase pathway dynamics. They aim to understand how the spacing of different colonic cell types and their communication are regulated. They found that apoptosis induces Erk signaling waves that prime cell movement and are essential to maintain tissue patterning in the organoid monolayers. The work presented here is of importance to the field and provides insights into how Erk waves driven by apoptosis can help maintain gut homeostasis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    The authors here follow-up on roles for signaling pathways like ERK in epithelial patterning that have been studied in an emerging literature in both, broadly, the cell competition field and, more specifically, in mouse intestinal organoids. They employ timelapse microscopy to study behavior of human colonic organoids in monolayers as the organoids initially self-organize. They then follow maintenance of organization into densely clustered nodes that have increased cells in cell cycle and the remaining more sparsely populated regions with fewer cycling cells. Nodes also show markers of in vivo colonic stem cells (Lgr5 and myc). They follow propagation of ERK waves using a genetic tool (ERK-JTR) and show that they can emerge from single apoptotic cells in between nodes.

    Strengths of the study include novelty of showing self-organization and behavior of human organoids over time, with good resolution, using microscopy, as well as sophisticated analysis techniques to interpret and present cumulative data over many experiments. Additionally, the paper adds important pieces of the puzzle with respect to how cells may compete and respond across an entire monolayer, and the tools and approaches lend themselves to studying many genes and signaling pathways besides simply Wnt vs ERK.

    Weaknesses in the current version of the manuscript:

    1. The manuscript is focused nearly exclusively on ERK and Wnt but not in terms of the broader context of interpretation of the response of a monolayer to apoptosis of single cells. Some of the original work in the field showed that apoptotic cells enacted Rho- and MLCK-dependent actomyosin contractility, which was proposed to signal neighboring cells by initially pulling them inwards via the contraction (PMIDS: 9456322, 10459006, 11283606, 21721944). But a more intestine-specific literature has long-been extant following up on the critical role of ROCK and MLCK in maintaining barrier after specifically intestinal-cell apoptosis (15825080, 21237166).

    -- A suggestion would be 1) to cite the relevant literature and 2) to interpret some of the experiments within the cytoskeletal mechanistic context already known. In addition to comments about PMA and ERK activation (see next point), the authors could test whether the ERK waves cause myosin II activation and/or are ROCK/MLCK-dependent. Given ROCK inhibition is frequently used in organoid culture, this would seem an obvious avenue to explore. Does the ERK wave propagate the cytoskeletal changes to close the gap and increase centrifugal motility and/or conversely does the actomyosin tugging of the apoptotic cell trigger ERK activation? (admittedly, the latter question may be hard to address). In short, there is a lot known about monolayer behavior in terms of dynamic cytoskeletal changes that can be addressed here to integrate with the Wnt/ERK roles.

    1. The authors use only PMA as an ERK activator. PMA is a broadly acting drug, principally known as a PI3K inducer. Obviously, Akt and other downstream action of PI3K means many other pathways are stimulated besides ERK. Indeed, ROCK and Src and other cytoskeleton-modifying pathways are modulated by PMA that may not correlate with the ERK effects. Additionally, the movies showing the effects of PMA treatment show a striking increase in apoptotic cells throughout the field, which would obviously confound the interpretation of what happens after relatively rare, internodal apoptotic cells die

    -- A strong suggestion would be to increase the routes to ERK activation the authors use. This could be via receptor tyrosine kinase stimulation (again, like ROCK, EGF is a key organoid medium component), though obviously that would not be much more specific than PMA, but the authors use EGFr inhibition to block ERK, so wouldn't stimulation be an apt converse approach? Genetic constitutively active KRAS might be introduced. Alternatively, there are pharmacological ways to increase pERK dramatically by inhibiting the dual action phosphatase (see eg PMID: 30475204 in a previous eLife paper). At the least, it would seem the authors should not use an approach that increases apoptosis dramatically.

    1. The movies clearly show many dividing cells that are between nodes, and they show apoptotic cells within nodes (eg movie 3a towards the end). While it's clear that apoptotic cells in internodal regions can elicit the wave behavior, it would seem that apoptosis does not universally do this, given the counter-examples.

    -- It would help if the authors could speak to this. Namely, in what cases are there no waves after apoptosis and what are the factors that might contribute (nearness to a node? nearness in time and space to another apoptotic cell?). Presumably, the events are relatively stochastic so there would be occasions for non-stereotypical behavior like wave front interference or augmentation in the case of closely located apoptotic cells.

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  3. Reviewer #2 (Public Review):

    The work by Pond, et al., uses patient derived organoid monolayers to interrogate MAPK signaling in real-time using an ERK reporter. This technology was developed previously to use a target domain of ERK that responds to phosphorylation by altering nuclear-cytoplasmic localization. The active ERK kinase can be inferred by cytoplasmic localization of the reporter. The premise of the paper is that this reporter can be used in human organoid cultures to understand ERK signaling dynamics. Figures 1 and 2 demonstrate the monolayer culture properties and how stem-like and differentiated domains for within the cultures, validated using RNA FISH for MYC, LGR5, and KRT20. Figure 3 describes how an ERK wave radiates out from an apoptotic cell in the cultures, and that the living cells migrate towards to dying cell, presumably to sustain a barrier. In figure 4, data is presented showing that PMA-mediated activation of ERK disrupts the patterning of the monolayers, dispersing the nodes of cells associated with stem/proliferative identity. Finally, in figure 5, the authors show that treating cultures with Wnt3a suppresses ERK activity, while inhibiting ERK may expand WNT/stem cells in the cultures.

    The study is interesting and the model system has a lot of potential.
    However, there are some concerns about the novelty. The reasons for this are:

    1 - the monolayer system has been demonstrated before, very nicely in a 2018 Dev. Cell paper from the Altschuler lab and one of the current manuscript authors.

    2 - ERK-KTR reporters have been used to demonstrate apoptosis induced signaling waves in the epithelium (Gagliardi, 2021, Dev. Cell.)

    3 - ERK activity suppressing stem cell fate has been documented previously (Riemer, 2015; Leach, 2021; Reischmann, 2020; Tong, 2017)

    So while there are exciting aspects of the work, including use of human tissues and live imaging of pathway dynamics, I feel that the novel discoveries using these technologies are somewhat limited.

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