Minimal essential requirements for neural tube self-organisation

Background

Turing wrote of embryonic development: “Most of an organism, most of the time, is developing from one pattern into another, rather than from homogeneity into a pattern” (Turing, 1952). However, this is not the case for many in vitro stem cell-derived systems, which have the uncanny ability to spontaneously break symmetry from homogeneous populations and generate precise spatial patterns of cell types (Ishihara and Tanaka, 2018). Such self-organization may enable biological systems to develop autonomously through intercellular interactions and to dynamically self-correct until a desired outcome is achieved (Ramos et al., 2024).

Neural tube organoids are remarkable in this sense: upon addition of a single inductive cue (retinoic acid), a single cell can proliferate and spontaneously break symmetry into two separate patterned populations, the floorplate precursors (FP) and early neural progenitors (eNP) (Meinhardt et al., 2014). The proportions of these two fates is remarkably conserved between different organoids, with FP cells composing around 25% of each organoid (Figure 1). This suggests that the proportional representation of the different cell types is somehow encoded in the system. In this preprint, the authors shed light on the underlying mechanisms of this symmetry breaking event and the subsequent establishment of robust cell type proportions.

Key findings

1. Symmetry breaking occurs through a transient progenitor state co-expressing two antagonistic master regulators of the two cell fates

Through a combination of timecourse scRNA-seq and flow cytometry, the authors identified a cell state that sits at the “head” of the bifurcating fate trajectory and can also be identified in the developing mouse neural tube in vivo. This state is characterised by co-expression of Foxa2 and Pax6, markers usually associated with FP and eNP fates respectively. The authors validate the mutual antagonising effect of these two transcription factors for pushing the cells towards one or the other fate.

2. Cell type proportions are dynamically allocated through population sensing

The authors observed that the cell fate choices of these “head” cells evolved over time: initially, they mostly contributed towards FPs, then they shifted towards eNPs. This suggests that cell fate allocation may be influenced by some kind of feedback, allowing the system to evolve towards a specific encoded cell type proportion.

To test this idea, the authors generated chimeric organoids by mixing WT cells with Foxa2 knockout cells, which are incapable of generating FP cells (Figure 2). If the cell fate choice was purely genetically encoded as a probabilistic outcome, the WT cells would always produce the same proportion of cell fate outcomes regardless of the genetic composition of the organoid. Instead, WT cells compensated for increasing numbers of Foxa2 knockout cells by proportionally producing more Foxa2-positive FPs, demonstrating that there is population-level sensing. The authors termed this process ‘regulative feedback’.

3. Identifying the signals regulating symmetry breaking and cell type proportion allocation

Interestingly, the final proportion of FPs and eNPs was largely insensitive to both the concentration and duration of the initial signalling cue, retinoic acid. This strengthens the notion that symmetry breaking and proportional cell fate allocation are intrinsically regulated. Consistently, inducing transient co-expression of Foxa2 and Pax6 was sufficient to bypass this inductive cue and initiate the self-organization program.

The authors propose BMP signalling as an important player in the regulative feedback, as shown by the increased proportions of FPs upon BMP inhibition. They suggest that emerging FPs activate BMP signalling, which biases the specification of “head” cells towards eNPs; thus, as FPs accumulate, they increasingly favour the other fate, creating a self-regulating mechanism that stabilises cell type proportions across organoids.

Why I chose this preprint

Symmetry breaking and self-organisation of specific cell type proportions are fascinating properties of developmental systems. To me, it is incredible that a single cell in a dish, given the right cue, can initiate such programs and give rise to reproducible patterns of cell types. What I found compelling in this preprint is the back-and-forth between modeling and experimental data  – particularily how time-course experiments informed the dynamical model, and how genetic perturbations served to challenge it.

Their model, which they term ‘regulative feedback’, has the advantage of being dynamic and self-regulating, enabling self-organisation of reproducible cell type proportions in vitro, which is particularly important for organoid models to reach their full potential in disease modelling and drug screening. Previous studies had identified other factors able to break symmetry in organoids, such as mechanical sensing and stochastic signalling heterogeneity (Gjorevski et al., 2022; McNamara et al., 2024). The extent to which regulative feedback is implicated in other developmental systems remains to be explored.

Questions for the authors

1. Would a regulative feedback mechanism be dosage-sensitive to BMP activation? If this is the case, is this surprising, given the robustness of the resulting cell type proportions?
2. Do you think regulative feedback plays a role not only in initially allocating cell type proportions but also dynamically maintaining them despite cell proliferation?
3. Could there be a spatial component to regulative feedback, potentially linked to your previous observations of BMP-mediated cell competition and cell sorting (Krammer et al., 2024)?
4. If self-organizing regulative feedback also operates in vivo, how could it be coordinated with external positional cues to ensure consistent ventral positioning of the floorplate across the neural tube?

References

Gjorevski, N., Nikolaev, M., Brown, T.E., Mitrofanova, O., Brandenberg, N., DelRio, F.W., Yavitt, F.M., Liberali, P., Anseth, K.S., Lutolf, M.P., 2022. Tissue geometry drives deterministic organoid patterning. Science 375, eaaw9021.

Ishihara, K., Tanaka, E.M., 2018. Spontaneous symmetry breaking and pattern formation of organoids. Current Opinion in Systems Biology, Development and differentiation 11, 123–128.

Krammer, T., Stuart, H.T., Gromberg, E., Ishihara, K., Cislo, D., Melchionda, M., Perez, F.B., Wang, J., Costantini, E., Lehr, S., Arbanas, L., Hörmann, A., Neumüller, R.A., Elvassore, N., Siggia, E., Briscoe, J., Kicheva, A., Tanaka, E.M., 2024. Mouse neural tube organoids self-organize floorplate through BMP-mediated cluster competition. Developmental Cell 59, 1940-1953.e10.

McNamara, H.M., Solley, S.C., Adamson, B., Chan, M.M., Toettcher, J.E., 2024. Recording morphogen signals reveals mechanisms underlying gastruloid symmetry breaking. Nat Cell Biol 26, 1832–1844.

Meinhardt, A., Eberle, D., Tazaki, A., Ranga, A., Niesche, M., Wilsch-Bräuninger, M., Stec, A., Schackert, G., Lutolf, M., Tanaka, E.M., 2014. 3D reconstitution of the patterned neural tube from embryonic stem cells. Stem Cell Reports 3, 987–999.

Ramos, R., Swedlund, B., Ganesan, A.K., Morsut, L., Maini, P.K., Monuki, E.S., Lander, A.D., Chuong, C.-M., Plikus, M.V., 2024. Parsing patterns: Emerging roles of tissue self-organization in health and disease. Cell 187, 3165–3186.

Turing, A.M., 1952. The chemical basis of morphogenesis. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 237, 37–72.

Tags: cell fate decisions, neural tube organoids, stem cells, symmetry breaking

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