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Summary and Overall Assessment

This paper investigates the mechanisms driving intestinal organoid morphogenesis. The bistable state where crypt cells can switch between two stable configurations, bulged (open) and budded (closed), based on mechanical and morphological history, is proposed as a crucial factor for robust development of organoid structures. The study integrates experimental observations from microscopy with theoretical predictions to explore how feedback mechanisms contribute to the establishment and maintenance of complex intestinal organoid morphology. Thus, through a combination of experimental and computational methods, a mechanistic biophysical picture of intestinal organoid formation is achieved, which is a useful contribution to the field of developmental biology. A few aspects need further clarification, particularly regarding data interpretation and outlook.

Overall, the study provides significant mechanical insights into complex intestinal crypt-villus organoid patterning. The integration of experimental and theoretical approaches is a notable strength, offering a novel framework for understanding organoid development. the paper would benefit from a clearer discussion of biological implications, and a nuanced presentation of conclusions.

Major Points

1.    Detailed Methodology

• Is there a measure for hysteresis in the model beyond being “qualitatively consistent” with the data? The support given for the statement that “ultimate morphological outcome depends on the history of the system” is based on volume change, but what are the overarching indicators that the system retains a shape memory? Is there a more concrete analytical connection between volume and morphology, e.g, calculation of a shape index? “Morphometric parameters” are mentioned in the discussion, data analysis, and supplementary section but not alluded to in the text. How were these used to inform model constraints?

• Given that adhesive interaction is accounted for in the surface tension term, what is the role of adhesion in affecting crypt geometry and lumen volume? Was this a consideration during experimental validation of differential tension modelling assumptions?

• What are the limitations of this experimental system in capturing physiologically relevant patterns? Comparison of organoid morphogenetic properties to patient-derived or explant specimens would be useful.

2.     Data Interpretation and Context

• It is essential to contextualize the findings within the larger framework of gut development and morphogenesis, explaining how they advance current knowledge in the field. The results effectively demonstrate a role for mechanical bistability during intestinal lumen formation, but the unified biological view presented in the abstract that “bistability arising from feedback between cellular tensions and fluid pressure could be a general mechanism to allow for the coordination of multicellular shape changes in developing systems” should be expounded upon. Is this generalization specific to organoid systems?

• What is the connection between stem cell fate differentiation and lumen which led to the original assumption that bistability would “resolve the experimental paradox of lumen inflation”?

• Are the “pharmacological experiments” mentioned in the abstract referring to PGE treatment? Perhaps this general statement should be clarified to “performed mechanical and pharmacological experiments to validate the key modelling assumptions and make quantitative predictions on organoid morphogenesis.”

3.     Clarifications Needed

• In the abstract, it is confusing that ‘direct mechanical’ is considered passive while ‘indirect mechanosensitive’ is considered active. This distinction should be clarified.

• The interpretation of the myosin intensity ratio is potentially problematic, as it is unclear how one can distinguish between simply more myosin at a location versus changes in tension.

• The link between mechanochemical signals and the resulting morphological changes needs clearer articulation. The paper would benefit from a detailed discussion on how these signals integrate at the cellular level to drive the observed structural outcomes.

Minor Points

1.     Improving Clarity

• A supplementary video showcasing the 3D vertex model would aid understanding about translation between experiment and theory.

• In Figure 1, it should be clearly stated in the caption that the arrows in 1A-C indicate the bud. The extent of the blue and orange curves in graphs 1A-C should be specified as standard deviations or confidence intervals. The meaning of "PGE treatment" mentioned in the caption needs to be clarified.

• In Figure 4D, the red/green images in the insets are difficult to discern. Adding annotations or arrows would help guide the reader.

2.     Consistent Terminology and Proofreading

• Uniform use of abbreviations and terminology throughout the manuscript will improve readability and reduce potential confusion. Through the paper, the word “mechanics” is connected (sometimes with a hyphen) to the word “chemical”, “osmotic”, “sensitive”, and “sensation”. Are these terms being used interchangeably? For example, after motivating the need for this study in the introduction comes the statement “here we propose a biophysical theory for the coordination of mechano-osmotic forces driving intestinal organoid morphogenesis.”

• The phrase “cytoskeletal tension” is mentioned only in the abstract. Perhaps it should also be referenced in relation to actomyosin within the main body of text.

3.     Literature Integration

• While the literature review is comprehensive, the paper could be improved by highlighting the novelty of its findings within a broader context of other organoid systems with highly folded complex geometries. Given that the model is specific to bulged versus budded crypt geometries, which principles can be extrapolated to other highly budded or branched systems e.g the drosophila gut (https://elifesciences.org/articles/77355) and mouse salivary gland (10.1016/j.cell.2021.05.015)?

Reviewer Expertise Statement This review is informed by expertise in high resolution optical imaging and computational modelling of embryonic systems.

Competing interests

The authors declare that they have no competing interests.