In preprints: exploring developmental robustness and timing with gastruloids

How developmental processes are coordinated in time and space to ensure the robust formation of complex, functional patterns during embryogenesis remains an outstanding question. Investigating this in the context of mammalian development poses a particular challenge, since the emergence of the body plan occurs within the uterine wall. Over the past decade, stem-cell-based models of mammalian development have been established as complementary systems to study some key aspects of mammalian embryogenesis in vitro (Martinez Arias et al., 2022; Cao et al., 2024; Shahbazi and Pasque, 2024). In addition to their accessibility and scalability, these models provide an exciting opportunity to study developmental processes in the absence of the constraints imposed by the in utero environment and the tightly controlled initial conditions that characterize development in vivo (Cao et al., 2024). Three recent preprints use gastruloids, a stem-cell-based embryo model that recapitulates some aspects of gastrulation, including the establishment of the germ layers and formation of the major body axes (Martinez Arias et al., 2022; Beccari et al., 2018; Veenvliet and Herrmann, 2021), to study the interplay between morphogenesis, transcriptional programs and physical parameters, under systematic variation of initial conditions (Bennabi et al., 2024 preprint; Fiuza et al., 2024 preprint; Oriola et al., 2024 preprint). This approach allows the authors to ‘decouple’ different effects, such as those of aggregate size, cell state heterogeneity and mechanics, on the dynamics and robustness of axial patterning and morphogenesis.One of the defining features of gastruloids as models of embryo development is their capacity to polarise and elongate along a self-organised anterior-posterior axis, concomitant with differentiation into the three germ layers (Martinez Arias et al., 2022; Beccari et al., 2018; Veenvliet and Herrmann, 2021). However, the propensity to successfully achieve such embryo-like features is variable, and seems to partially depend on the initial conditions, such as aggregate size, stem cell pluripotency conditions and culture medium composition (Anlaş et al., 2025 preprint; Blotenburg et al., 2025; Avni et al., 2023; Veenvliet et al., 2021).A recurrent finding in all three studies is the impact of initial aggregate size (as tuned by the number of cells seeded for aggregation) and/or the composition of the seeded cells on the timing of polarisation and elongation. By altering the initial cell number in their aggregates, Bennabi et al. and Fiuza et al. study the effect of initial size on developmental timing and gene expression patterns (Bennabi et al., 2024 preprint; Fiuza et al., 2024 preprint). Both studies find that, within a range of initial cell seeding numbers, gastruloids exhibit robust polarisation and elongation, as well as relatively conserved gene expression patterns, aligning with recent findings (Anlaş et al., 2024; Merle et al., 2024). However, the data of Bennabi and colleagues suggest that there is a temporal decoupling of morphogenesis from transcriptional programs, showing that, whereas gastruloids maintain stable cell fate compositions across a broad range of sizes, morphogenetic dynamics are highly size-dependent (Bennabi et al., 2024 preprint). Specifically, they find that larger gastruloids exhibit delayed symmetry breaking and prolonged axial elongation time scales, whereas smaller aggregates initiate elongation earlier but often collapse due to progenitor pool exhaustion (Bennabi et al., 2024 preprint).Furthermore, both studies show that outside a defined size range, deviations in cell composition and gene expression emerge: very small gastruloids exhibit a neural bias and fail to properly form paraxial mesoderm, while large aggregates struggle with coalescing multiple expression foci of Brachyury – a transcription factor that marks axial progenitors and their mesodermal progeny, and is crucial for axial elongation in embryos and gastruloids (Martinez Arias et al., 2022; Veenvliet and Herrmann, 2021; Wilkinson et al., 1990) – into a single posterior pole. These findings suggest a defined size range for robust patterning and morphogenesis in gastruloids, in agreement with previous studies (Anlaş et al., 2024; Boxman et al., 2016; van den Brink et al., 2014). Interestingly, this number is within the order of magnitude of epiblast cell numbers in E5.5-E6.5 mouse embryos (Snow, 1977).By either fusing together or cutting gastruloids prior to their elongation, Bennabi and colleagues tested the influence of absolute size versus initial cell number on morphogenetic timing and expression dynamics of important developmental marker genes. They found that the fusion of initially small gastruloids leads to behaviour typical of larger gastruloids (high initial cell number), whereas dividing large gastruloids into smaller fragments drives them to behave more similarly to originally small gastruloids (low initial cell seeding number). These results point towards an effect of absolute size, as well as a capacity for adaptation to size perturbations, during the self-organised axial patterning in gastruloids.Oriola and colleagues provide additional mechanistic insights into the onset of symmetry breaking and elongation in gastruloids by controlling the initial gastruloid cellular composition. During aggregate formation, they systematically change the proportion of cells expressing brachyury (Bra/T) and find a strong effect on the timing of symmetry breaking and the onset of elongation (Oriola et al., 2024 preprint). Based on these results and on mathematical modelling, they suggest a feedback mechanism where pluripotent cells inhibit mesoderm differentiation, thereby modulating the timing of symmetry breaking and the transition to axial elongation.The formation of a single anterior-posterior axis, and elongation along this axis, are some of the most universal characteristics of animal development (Boutillon et al., 2024). The ability of gastruloids to recapitulate this in the absence of external cues points towards the role of intrinsic self-organisation mechanisms in driving this process. However, axis formation in gastruloids is far from robust, with the occasional emergence of multi-axial morphologies observed across multiple studies (Anlaş et al., 2024; van den Brink et al., 2014). In the preprints by Bennabi et al. and Fiuza et al., the authors quantitatively investigate the formation of multi-axial morphologies as a function of aggregate size. Both studies find that the prevalence of multi-axial morphologies increases with aggregate size (controlled by initial cell seeding number). Interestingly, both studies find that the frequency of multi-polarity decreases with time, indicating the tendency of multiple poles to resolve into single ones (Bennabi et al., 2024 preprint; Fiuza et al., 2024 preprint). Further insight is gained by Bennabi and colleagues through live imaging of gastruloids marked with a fluorescent reporter for Mesp2 (expressed at the anterior), which enables them to follow the dynamics of multi-polarity over gastruloid development in detail. Within a broad size range, they find that the timing of resolution from multiple poles into a single pole is correlated with overall system size, and with the distance between the poles that resolve (Bennabi et al., 2024 preprint).This size-dependent occurrence, and subsequent resolution, of multi-polarity in gastruloids is reminiscent of that observed in other developmental systems, from the regeneration of Hydra cell aggregates (Sato and Sawada, 1989) to the formation of floorplate clusters in neural tube organoids (Krammer et al., 2024). A fascinating question in all these cases is the mechanism through which multi-polarity is resolved. In the case of the neural tube organoids, both cluster competition and sorting play a role (Krammer et al., 2024). Bennabi and colleagues show that, in gastruloids, cluster sorting (multiple poles coming together and converging) is likely involved, but other possibilities, such as mutual inhibition with no contact, cannot be ruled out (Bennabi et al., 2024 preprint). Furthermore, while multi-polarity is observed for both anterior and posterior poles, their resolution dynamics and driving mechanisms may differ. Interestingly, the results of both Fiuza et al. and Oriola et al. hint at a role for mechanics in the context of polarisation and/or the resolution of multi-polarity (see below) (Fiuza et al., 2024 preprint; Oriola et al., 2024 preprint). It remains an outstanding question whether, and to what extent, the mechanisms that appear to support the robustness of axis formation in gastruloids are similar to those operating in vivo.The potential role for mechanics in directing and regulating morphogenesis across scales is becoming increasingly appreciated. The possibility of mechanical properties playing a role in gastruloid development is explored in the preprints by Oriola et al. and Fiuza et al., who employ tissue rheology and aggregate fusion assays to investigate the effect of cell identity (T− versus T+) and aggregate size on mechanical properties (Fiuza et al., 2024 preprint; Oriola et al., 2024 preprint). Their findings indicate that while the (inferred) material properties do not depend on aggregate size, they are markedly affected by the proportion of T+ cells. This was true both when comparing different initial cell compositions (T− versus T+) and when comparing later timepoints (characterised by a higher proportion of T+ cells) with earlier ones. These results suggest that, in aggregates containing both populations, this difference in mechanical properties could drive cell sorting and the polarisation of the aggregates, and possibly play a role in the resolution of multi-polarity. Both studies also suggest that differences in adhesion between T+ and T− cells may be related to the observed mechanical differences. These findings align with previous work on cell sorting during gastruloid symmetry breaking based on differential E-cadherin expression (Hashmi et al., 2022), and differences in surface tension between T+ and T− cell populations contributing to symmetry breaking and axis formation (Gsell et al., 2025).All three preprints demonstrate the power of bottom-up stem-cell-based embryo models, such as gastruloids, to systematically test how parameters that are highly constrained during in vivo development affect developmental processes (Cao et al., 2024). Collectively, the preprints highlight the robustness of gene expression programs to modulations of these parameters, while also revealing the limits to this robustness, as well as the strong effects of initial conditions on timing and physical organisation. Some fascinating open questions are raised: How is robustness of transcriptional programs and cell fate composition achieved across a broad range of sizes? Why does this self-regulation break down at the extremes? And, of course, how do these findings translate to in vivo embryogenesis, where size regulation mechanisms are in place?Over four decades ago, Lewis and Rossant created double-sized mouse embryos by fusion, and found that size regulation happens between implantation and gastrulation, by adjusting cell cycle length (Lewis and Rossant, 1982). Why such in vivo checkpoints are only partially in place in gastruloids, and how this relates to the presence or absence of morphogenetic constraints and other, possibly redundant, control mechanisms that govern robustness of axial morphogenesis and patterning, remains a largely unanswered question. The accessibility, tractability and scalability of stem cell-based embryo models provide an exciting possibility to start shedding light on these fascinating questions.We thank all members of the Veenvliet lab for discussions on the preprints.